Phytases, nucleic acids encoding them and methods for making and using them

ABSTRACT

This invention relates to phytases, polynucleotides encoding them, uses of the polynucleotides and polypeptides of the invention, as well as the production and isolation of such polynucleotides and polypeptides. In particular, the invention provides polypeptides having phytase activity under high temperature conditions, and phytases that retain activity after exposure to high temperatures. The invention further provides phytases which have increased gastric lability. The phytases of the invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients. The phytases of the invention can be formulated as foods or feeds or supplements for either to, e.g., aid in the digestion of phytate.

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Provisional Application Ser. No. (“USSN”) 61/180,283, filed May21, 2009. The aforementioned application is explicitly incorporatedherein by reference in its entirety and for all purposes.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application is being filed electronically via the USPTO EFS-WEBserver, as authorized and set forth in MPEP §1730 II.B.2(a)(A), and thiselectronic filing includes an electronically submitted sequence (SEQ ID)listing. The entire content of this sequence listing is hereinincorporated by reference for all purposes. The sequence listing isidentified on the electronically filed .txt file as follows:

File Name Date of Creation Size (bytes) D1370_17WO_sequence_listing.txtMay 20, 2010 58,575 bytes

FIELD OF THE INVENTION

This invention relates to phytases, polynucleotides encoding them, usesof the polynucleotides and polypeptides of the invention, as well as theproduction and isolation of such polynucleotides and polypeptides. Inparticular, the invention provides polypeptides having phytase activityunder high temperature conditions, and phytases that retain activityafter exposure to high temperatures. The invention further providesphytases which have increased gastric lability. The phytases of theinvention can be used in foodstuffs to improve the feeding value ofphytate rich ingredients. The phytases of the invention can beformulated as foods or feeds or supplements for either to, e.g., aid inthe digestion of phytate. The foods or feeds of the invention can be inthe form of pellets, liquids, powders and the like. In one aspect,phytases of the invention are stabile against thermal denaturationduring pelleting; and this decreases the cost of the phytase productwhile maintaining in vivo efficacy and detection of activity in feed.

BACKGROUND

Minerals are essential elements for the growth of all organisms. Dietaryminerals can be derived from many source materials, including plants.For example, plant seeds are a rich source of minerals since theycontain ions that are complexed with the phosphate groups of phytic acidmolecules. These phytate-associated minerals may, in some cases, meetthe dietary needs of some species of farmed organisms, such asmulti-stomached ruminants. Accordingly, in some cases ruminants requireless dietary supplementation with inorganic phosphate and mineralsbecause microorganisms in the rumen produce enzymes that catalyzeconversion of phytate (myo-inositol-hexaphosphate) to inositol andinorganic phosphate. In the process, minerals that have been complexedwith phytate are released. The majority of species of farmed organisms,however, are unable to efficiently utilize phytate-associated minerals.Thus, for example, in the livestock production of monogastric animals(e.g., pigs, birds, and fish), feed is commonly supplemented withminerals and/or with antibiotic substances that alter the digestiveflora environment of the consuming organism to enhance growth rates.

As such, there are many problematic burdens—related to nutrition, exvivo processing steps, health and medicine, environmental conservation,and resource management—that are associated with an insufficienthydrolysis of phytate in many applications. The following arenon-limiting examples of these problems:

-   -   1) The supplementation of diets with inorganic minerals is a        costly expense.    -   2) The presence of unhydrolyzed phytate is undesirable and        problematic in many ex vivo applications (e.g. by causing the        presence of unwanted sludge).    -   3) The supplementation of diets with antibiotics poses a medical        threat to humans and animals alike by increasing the abundance        of antibiotic-tolerant pathogens.    -   4) The discharge of unabsorbed fecal minerals into the        environment disrupts and damages the ecosystems of surrounding        soils, fish farm waters, and surface waters at large.    -   5) The valuable nutritional offerings of many potential        foodstuffs remain significantly untapped and squandered.

Consequently, phytate-containing foodstuffs require supplementation withexogenous nutrients and/or with a source of phytase activity in order toamend their deficient nutritional offerings upon consumption by a verylarge number of species of organisms.

Consequently, there is a need for means to achieve efficient and costeffective hydrolysis of phytate in various applications. Particularly,there is a need for means to optimize the hydrolysis of phytate incommercial applications. In a particular aspect, there is a need tooptimize commercial treatment methods that improve the nutritionalofferings of phytate-containing foodstuffs for consumption by humans andfarmed animals.

SUMMARY OF THE INVENTION

This invention provides polypeptides having phytase activity,polynucleotides encoding them, uses of the polynucleotides andpolypeptides of the invention, and methods for the production andisolation of such polynucleotides and polypeptides. In one aspect, theinvention provides polypeptides having phytase activity under hightemperature conditions, and phytases that retain activity after exposureto high temperatures. The phytases of the invention can be used infoodstuffs to improve the feeding value of phytate rich ingredients. Thephytases of the invention can be formulated as foods or feeds orsupplements for either to, e.g., aid in the digestion of phytate. Thefoods or feeds of the invention can be in the form of pellets, tablets,pills, liquids, powders, sprays and the like. In one aspect, phytases ofthe invention are stabile against thermal denaturation during pelleting;and this decreases the cost of the phytase product while maintaining invivo efficacy and detection of activity in feed.

SUMMARY

The invention provides isolated, synthetic, or recombinant nucleic acidscomprising

(a) (i) a nucleic acid sequence encoding a polypeptide having a phytaseactivity and having at least 95%, 96% 97%, 98% or 99% or more sequenceidentity to SEQ ID NO:1, wherein the polypeptide comprises at least onemutation listed in Table 4, 5, 6, 7, 9, or any combination thereof;

(ii) a polynucleotide encoding a polypeptide having at least 95%, 96%97%, 98% or 99% or more sequence identity to SEQ ID NO:2, wherein thepolypeptide comprises at least one mutation listed in Table 4, 5, 6, 7,9, or any combination thereof; or

(a) (i) a nucleic acid sequence encoding a polypeptide having a phytaseactivity and having at least 95%, 96% 97%, 98% or 99% or more sequenceidentity to SEQ ID NO:1, wherein the polypeptide comprises at least onemutation listed in Table 4, 5, 6, 7, 9, or any combination thereof;

(ii) a polynucleotide encoding a polypeptide having at least 95%, 96%97%, 98% or 99% or more sequence identity to SEQ ID NO:2, wherein thepolypeptide comprises at least one mutation listed in Table 4, 5, 6, 7,9, or any combination thereof; or

(iii) the nucleic acid sequence of (i) or (ii), wherein the sequenceidentities are determined by analysis with a sequence comparisonalgorithm or by a visual inspection, and optionally the sequencecomparison algorithm is a BLAST version 2.2.2 algorithm where afiltering setting is set to blastall -p blastp -d “nr pataa” -F F, andall other options are set to default;

(b) the nucleic acid of (a), wherein the at least one mutation is:A109V, A232P, A236H, A236T, A248L, A248T, A274F, A274I, A274L, A274T,A274V, A429P, C155Y, D139Y, E113P, F147Y, F194L, G171M, G171S, G257A,G257R, G353C, G395E, G395I, G395L, G395Q, G395T, G67A, H263P, H272W,I107H, I107P, I108A, I108Q, I108R, I108S, I108Y, I174F, I174P, I427G,I427S, I427T, K151H, K151P, L126R, L146R, L146T, L150T, L150Y, L157C,L157P, L167S, L192F, L216T, L235I, L244S, L269I, L269T, L296T, L379S,L379V, L50W, M51A, M51G, M51L, N148K, N148M, N148R, N161K, N266P, N339E,N348K, N348W, P100A, P145L, P149L, P149N, P217D, P217G, P217L, P217S,P254S, P269L, P343E, P343I, P343L, P343N, P343R, P343V, Q137F, Q137L,Q137V, Q137Y, Q246W, Q247H, Q275H, Q309P, Q377R, Q381S, Q86H, S102A,S102Y, S173G, S173H, S173V, S197G, S208P, S211H, S218I, S218Y, S389H,S389V, T163P, T282H, T291V, T291W, T341D, T48F, T48H, T48I, T48K, T48L,T48M, T48V, T48W, T48Y, V162L, V162T, V191A, V422M, W265L, Y79H, Y79N,Y79S, or Y79W;

(c) the nucleic acid of (b), wherein the polypeptide further comprisesat least one mutation of: C226D, D164R, G179R, N159V, Q275V, T163R, orT349Y; or (d) sequences fully complementary thereto. All of thesenucleic acids are “nucleic acids of the invention”, encoding“polypeptides of the invention”.

In one aspect, the sequence comparison algorithm is a BLAST version2.2.2 algorithm where a filtering setting is set to blastall -p blastp-d “nr pataa” -F F, and all other options are set to default.

In one aspect, the nucleic acid sequences of the invention lack ahomologous nucleic acid sequence encoding a signal sequence, proproteinsequence, or promoter sequence. In another aspect, the nucleic acids ofthe invention further comprise of a heterologous nucleic acid sequence,wherein optionally the heterologous nucleic acid sequence comprises, orconsists of a sequence encoding a heterologous signal sequence, a tag,an epitope, or a promoter sequence. In another aspect, the heterologousnucleic acid sequence encodes a heterologous signal sequence comprisingor consisting of an N-terminal and/or C-terminal extension for targetingto an endoplasmic reticulum (ER) or endomembrane, or to a plantendoplasmic reticulum (ER) or endomembrane system, or the heterologoussequence encodes a restriction site. In yet another aspect, theheterologous promoter sequence comprises or consists of a constitutiveor inducible promoter, or a cell type specific promoter, or a plantspecific promoter, or a maize specific promoter.

In one aspect, the phytase activity comprises catalysis of phytate(myo-inositol-hexaphosphate) to inositol and inorganic phosphate; or,the hydrolysis of phytate (myo-inositol-hexaphosphate). In anotheraspect, the phytase activity comprises catalyzing hydrolysis of aphytate in a feed, a food product or a beverage, or a feed, food productor beverage comprising a cereal-based animal feed, a wort or a beer, adough, a fruit or a vegetable; or, catalyzing hydrolysis of a phytate ina microbial cell, a fungal cell, a mammalian cell or a plant cell.

In one aspect, the phytases of the invention are thermotolerant, andoptionally the polypeptide retains a phytase activity after exposure toa temperature in the range from about −100° C. to about −80° C., about−80° C. to about −40° C., about −40° C. to about −20° C., about −20° C.to about 0° C., about 0° C. to about 37° C., about 0° C. to about 5° C.,about 5° C. to about 15° C., about 15° C. to about 25° C., about 25° C.to about 37° C., about 37° C. to about 45° C., about 45° C. to about 55°C., about 55° C. to about 70° C., about 70° C. to about 75° C., about75° C. to about 85° C., about 85° C. to about 90° C., about 90° C. toabout 95° C., about 95° C. to about 100° C., about 100° C. to about 105°C., about 105° C. to about 110° C., about 110° C. to about 120° C., or95° C., 96° C., 97° C., 98° C., 99° C., 100° C., 101° C., 102° C., 103°C., 104° C., 105° C., 106° C., 107° C., 108° C., 109° C., 110° C., 111°C., 112° C., 113° C., 114° C., 115° C. or more. In one aspect, thephytases of the invention are thermostable, and optionally the phytaseretains activity at a temperature in the range from about −100° C. toabout −80° C., about −80° C. to about −40° C., about −40° C. to about−20° C., about −20° C. to about 0° C., about 0° C. to about 37° C.,about 0° C. to about 5° C., about 5° C. to about 15° C., about 15° C. toabout 25° C., about 25° C. to about 37° C., about 37° C. to about 45°C., about 45° C. to about 55° C., about 55° C. to about 70° C., about70° C. to about 75° C., about 75° C. to about 85° C., about 85° C. toabout 90° C., about 90° C. to about 95° C., about 95° C. to about 100°C., about 100° C. to about 105° C., about 105° C. to about 110° C.,about 110° C. to about 120° C., or 95° C., 96° C., 97° C., 98° C., 99°C., 100° C., 101° C., 102° C., 103° C., 104° C., 105° C., 106° C., 107°C., 108° C., 109° C., 110° C., 111° C., 112° C., 113° C., 114° C., 115°C. or more.

In another embodiment, the phytases of the inventions are thermotolerantor thermoactive at an acidic pH of about pH 6.5, pH 6, pH 5.5, pH 5, pH4.5, pH 4.0, pH 3.5, pH 3.0 or less, or the phytase polypeptide isthermotolerant or thermoactive at about pH 7, pH 7.5, pH 8.0, pH 8.5, pH9, pH 9.5, pH 10, pH 10.5, pH 11.0, pH 11.5, pH 12.0, pH 12.5 or more.

The invention provides expression cassettes, vectors, cloning vehicles,expression vectors, and cloning vectors comprising a nucleic acid of theinvention, or having contained therein a nucleic acid of the invention(which include nucleic acids encoding polypeptides of the invention),wherein optionally the expression cassette, cloning vehicle or vectorcomprises or is a viral vector, a plasmid, a phage, a phagemid, acosmid, a fosmid, a bacteriophage or an artificial chromosome, the viralvector comprises or is an adenovirus vector, a retroviral vector or anadeno-associated viral vector, or the expression cassette, cloningvehicle or vector comprises or is a bacterial artificial chromosome(BAC), a bacteriophage P1-derived vector (PAC), a yeast artificialchromosome (YAC) or a mammalian artificial chromosome (MAC).

The invention provides transformed cells, transduced cells, host cellsand the like comprising a nucleic acid of the invention, or havingcontained therein a nucleic acid of the invention (which include nucleicacids encoding polypeptides of the invention), or the expressioncassette, vector, cloning vehicle, expression vector, or cloning vectorof the invention, wherein optionally the cell is a bacterial cell, amammalian cell, a fungal cell, a yeast cell, an insect cell or a plantcell.

The invention provides transgenic non-human animals comprising a nucleicacid of the invention, or having contained therein a nucleic acid of theinvention (which include nucleic acids encoding polypeptides of theinvention), or the expression cassette, vector, cloning vehicle,expression vector, or cloning vector of the invention, whereinoptionally the animal is a mouse, a rat, a goat, a rabbit, a sheep, apig or a cow.

The invention provides transgenic plants (including plant parts, e.g.,processed, or harvested, e.g., leaves, stems, roots or fruits) or seedscomprising a nucleic acid of the invention, or having contained thereina nucleic acid of the invention (which include nucleic acids encodingpolypeptides of the invention), or the expression cassette, vector,cloning vehicle, expression vector, or cloning vector of the invention,wherein optionally the plant is a corn plant, a potato plant, a tomatoplant, a wheat plant, an oilseed plant, a rapeseed plant, a soybeanplant or a tobacco plant, and optionally the seed is a corn seed, awheat kernel, an oilseed, a rapeseed, a soybean seed, a palm kernel, asunflower seed, a sesame seed, a peanut or a tobacco plant seed. Inalternative embodiments, the plant or a seed produced from a seed orplant of the invention, or a plant or seed of the invention, can includecrop plants, for example, corn, alfalfa, sunflower, Brassica, soybean,sugar cane, cotton, safflower, peanut, sorghum, wheat, oat, rye, millet,barley, rice, conifers, legume crops, e.g., pea, bean and soybean,starchy tuber/roots, e.g., potato, sweet potato, cassava, taro, cannaand sugar beet and the like, or the plant can be a corn plant, a potatoplant, a tomato plant, a wheat plant, an oilseed plant, a rapeseedplant, a soybean plant or a tobacco plant, or a forage and/or feed plantfor an animal, or for a ruminant animal, or the plant can be orcomprises the forage or feed plant is hay, corn, millet, soy, wheat,buckwheat, barley, alfalfa, rye, an annual grass, sorghum, sudangrass,veldt grass or buffet grass; or, the seed is a corn seed, a wheatkernel, an oilseed, a rapeseed, a soybean seed, a palm kernel, asunflower seed, a sesame seed, a peanut or peanut seed, an alfalfa seed,a cotton seed, a safflower seed, a sorghum seed, an oat kernel, a ryeseed, a millet seed, a barley seed, a rice kernel, a pea seed, or atobacco plant seed, or the plant is corn, alfalfa, sunflower, Brassica,soybean, sugar cane, cotton, safflower, peanut, sorghum, wheat, oat,rye, millet, barley, rice, conifers, pea, bean, soybean, potato, sweetpotato, cassava, taro, canna or sugar beet.

The invention provides antisense oligonucleotides comprising a nucleicacid which is antisense to a nucleic acid of the invention and encodingat least one mutation listed in Table 4, 5, 6, 7, 9, or any combinationthereof, wherein optionally the antisense oligonucleotide is betweenabout 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, about 60to 100 or about 50 to 150 bases in length. The invention also providesribozymes and/or iRNA (e.g., siRNA or miRNA) comprising antisensesequences of the invention.

The invention provides methods of inhibiting the translation of aphytase message in a cell comprising administering to the cell orexpressing in the cell an antisense oligonucleotide of the invention.

The invention provides isolated, synthetic, or recombinant polypeptidescomprising

(a) (i) an amino acid sequence encoded by the nucleic acids of theinvention;

(ii) an amino acid sequence having at least 95%, 96% 97%, 98% or 99%sequence identity to SEQ ID NO:2, wherein the polypeptide comprises atleast one mutation listed in Table 4, 5, 6, 7, 9, or any combinationthereof; or

(iii) the polypeptide of (i) or (ii), wherein the sequence identitiesare determined by analysis with a sequence comparison algorithm or by avisual inspection;

(b) the polypeptide of (a), wherein the at least one mutation is: A109V,A232P, A236H, A236T, A248L, A248T, A274F, A274I, A274L, A274T, A274V,A429P, C155Y, D139Y, E113P, F147Y, F194L, G171M, G171S, G257A, G257R,G353C, G395E, G395I, G395L, G395Q, G395T, G67A, H263P, H272W, I107H,I107P, I108A, I108Q, I108R, I108S, I108Y, I174F, I174P, I427G, I427S,I427T, K151H, K151P, L126R, L146R, L146T, L150T, L150Y, L157C, L157P,L167S, L192F, L216T, L235I, L244S, L269I, L269T, L296T, L379S, L379V,L50W, M51A, M51G, M51L, N148K, N148M, N148R, N161K, N266P, N339E, N348K,N348W, P100A, P145L, P149L, P149N, P217D, P217G, P217L, P217S, P254S,P269L, P343E, P343I, P343L, P343N, P343R, P343V, Q137F, Q137L, Q137V,Q137Y, Q246W, Q247H, Q275H, Q309P, Q377R, Q381S, Q86H, S102A, S102Y,S173G, S173H, S173V, S197G, S208P, S211H, S218I, S218Y, S389H, S389V,T163P, T282H, T291V, T291W, T341D, T48F, T48H, T48I, T48K, T48L, T48M,T48V, T48W, T48Y, V162L, V162T, V191A, V422M, W265L, Y79H, Y79N, Y79S,or Y79W; or

(c) the polypeptide of (b), wherein the polypeptide further comprises atleast one mutation of: C226D, D164R, G179R, N159V, Q275V, T163R, orT349Y.

In one aspect, the phytase activity comprises catalysis of phytate(myo-inositol-hexaphosphate) to inositol and inorganic phosphate; or,the hydrolysis of phytate (myo-inositol-hexaphosphate). In anotheraspect, the phytase activity comprises catalyzing hydrolysis of aphytate in a feed, a food product or a beverage, or a feed, food productor beverage comprising a cereal-based animal feed, a wort or a beer, adough, a fruit or a vegetable; or, catalyzing hydrolysis of a phytate ina microbial cell, a fungal cell, a mammalian cell or a plant cell.

The invention provides polypeptides of the invention that have phytaseactivity whose activity is thermotolerant, and optionally thepolypeptide retains a phytase activity after exposure to a temperaturein the range of from about −100° C. to about −80° C., about −80° C. toabout −40° C., about −40° C. to about −20° C., about −20° C. to about 0°C., about 0° C. to about 5° C., about 5° C. to about 15° C., about 15°C. to about 25° C., about 25° C. to about 37° C., about 37° C. to about45° C., about 45° C. to about 55° C., about 55° C. to about 70° C.,about 70° C. to about 75° C., about 75° C. to about 85° C., about 85° C.to about 90° C., about 90° C. to about 95° C., about 95° C. to about100° C., about 100° C. to about 105° C., about 105° C. to about 110° C.,about 110° C. to about 120° C., or 95° C., 96° C., 97° C., 98° C., 99°C., 100° C., 101° C., 102° C., 103° C., 104° C., 105° C., 106° C., 107°C., 108° C., 109° C., 110° C., 111° C., 112° C., 113° C., 114° C., 115°C. or more. The thermotolerant polypeptides according to the inventioncan retain activity, e.g. a phytase activity, after exposure to atemperature in the range from about −100° C. to about −80° C., about−80° C. to about −40° C., about −40° C. to about −20° C., about −20° C.to about 0° C., about 0° C. to about 5° C., about 5° C. to about 15° C.,about 15° C. to about 25° C., about 25° C. to about 37° C., about 37° C.to about 45° C., about 45° C. to about 55° C., about 55° C. to about 70°C., about 70° C. to about 75° C., about 75° C. to about 85° C., about85° C. to about 90° C., about 90° C. to about 95° C., about 95° C. toabout 100° C., about 100° C. to about 105° C., about 105° C. to about110° C., about 110° C. to about 120° C., or 95° C., 96° C., 97° C., 98°C., 99° C., 100° C., 101° C., 102° C., 103° C., 104° C., 105° C., 106°C., 107° C., 108° C., 109° C., 110° C., 111° C., 112° C., 113° C., 114°C., 115° C. or more. In some embodiments, the thermotolerantpolypeptides according to the invention retains activity, e.g. a phytaseactivity, after exposure to a temperature in the ranges described above,at about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5.0,about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5,about pH 8.0, about pH 8.5, about pH 9.0, about pH 9.5, about pH 10.0,about pH 10.5, about pH 11.0, about pH 11.5, about pH 12.0 or more.

The invention provides polypeptides of the invention that have phytaseactivity whose activity is thermostable. For example, a polypeptide ofthe invention can be thermostable. The thermostable polypeptideaccording to the invention can retain binding and/or enzymatic activity,e.g., a phytase activity, under conditions comprising a temperaturerange from about −100° C. to about −80° C., about −80° C. to about −40°C., about −40° C. to about −20° C., about −20° C. to about 0° C., about0° C. to about 37° C., about 0° C. to about 5° C., about 5° C. to about15° C., about 15° C. to about 25° C., about 25° C. to about 37° C.,about 37° C. to about 45° C., about 45° C. to about 55° C., about 55° C.to about 70° C., about 70° C. to about 75° C., about 75° C. to about 85°C., about 85° C. to about 90° C., about 90° C. to about 95° C., about95° C. to about 100° C., about 100° C. to about 105° C., about 105° C.to about 110° C., about 110° C. to about 120° C., or 95° C., 96° C., 97°C., 98° C., 99° C., 100° C., 101° C., 102° C., 103° C., 104° C., 105°C., 106° C., 107° C., 108° C., 109° C., 110° C., 111° C., 112° C., 113°C., 114° C., 115° C. or more. The thermostable polypeptides according tothe invention can retain activity, e.g. a phytase activity, intemperatures in the range from about −100° C. to about −80° C., about−80° C. to about −40° C., about −40° C. to about −20° C., about −20° C.to about 0° C., about 0° C. to about 5° C., about 5° C. to about 15° C.,about 15° C. to about 25° C., about 25° C. to about 37° C., about 37° C.to about 45° C., about 45° C. to about 55° C., about 55° C. to about 70°C., about 70° C. to about 75° C., about 75° C. to about 85° C., about85° C. to about 90° C., about 90° C. to about 95° C., about 95° C. toabout 100° C., about 100° C. to about 105° C., about 105° C. to about110° C., about 110° C. to about 120° C., or 95° C., 96° C., 97° C., 98°C., 99° C., 100° C., 101° C., 102° C., 103° C., 104° C., 105° C., 106°C., 107° C., 108° C., 109° C., 110° C., 111° C., 112° C., 113° C., 114°C., 115° C. or more. In some embodiments, the thermostable polypeptidesaccording to the invention retains activity, e.g., a phytase activity,at a temperature in the ranges described above, at about pH 3.0, aboutpH 3.5, about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH8.5, about pH 9.0, about pH 9.5, about pH 10.0, about pH 10.5, about pH11.0, about pH 11.5, about pH 12.0 or more.

The invention provides isolated, synthetic or recombinant polypeptidescomprising an amino acid sequence of the invention and (a) lacking ahomologous signal sequence (leader peptide) or proprotein sequence; (b)lacking a signal sequence (leader peptide) and further comprising aheterologous signal sequence (leader peptide); (c) the amino acidsequence of (a) or (b) and further comprising a heterologous sequence,wherein optionally the heterologous sequence comprises, or consists of aheterologous signal sequence, or a tag or an epitope, or theheterologous sequence comprises an identification peptide. In oneaspect, the heterologous signal sequence comprises or consists of anN-terminal and/or C-terminal extension for targeting to an endoplasmicreticulum (ER) or endomembrane, or to a plant endoplasmic reticulum (ER)or endomembrane system, or the heterologous amino acid sequencecomprises, or consists of an enzyme target site. In another aspect, thepolypeptide of the invention further comprises additional amino acidresidues between a signal sequence (leader sequence or leader peptide)and the enzyme.

In one aspect, the phytase activity of any polypeptide of the inventioncomprises (has) a specific activity: at about 37° C. in the range fromabout 100 to about 1000 units per milligram of protein; or, from about500 to about 750 units per milligram of protein; or, at 37° C. in therange from about 500 to about 1200 units per milligram of protein; or,at 37° C. in the range from about 750 to about 1000 units per milligramof protein. In one aspect, the thermotolerant phytase activity comprisesa specific activity after exposure to a temperature at about 37° C. inthe range from about 100 to about 1000 units per milligram of protein;or, the thermostable phytase activity comprises a specific activity fromabout 500 to about 750 units per milligram of protein; or, thethermostable phytase activity comprises a specific activity at 37° C. inthe range from about 500 to about 1200 units per milligram of protein;or, the thermostable phytase activity comprises a specific activity at37° C. in the range from about 750 to about 1000 units per milligram ofprotein. In one aspect, the thermostable phytase activity comprises aspecific activity under conditions comprising a temperature of about 37°C. in the range from about 100 to about 1000 units per milligram ofprotein; or, the thermostable phytase activity comprises a specificactivity from about 500 to about 750 units per milligram of protein; or,the thermostable phytase activity comprises a specific activity at 37°C. in the range from about 500 to about 1200 units per milligram ofprotein; or, the thermostable phytase activity comprises a specificactivity at 37° C. in the range from about 750 to about 1000 units permilligram of protein.

In one aspect, a polypeptide of the invention is glycosylated orcomprises at least one glycosylation site, wherein optionally theglycosylation is an N-linked glycosylation, or an O-linkedglycosylation, and optionally the polypeptide is glycosylated afterbeing expressed in a yeast, which optionally is a P. pastoris or a S.pombe.

In one aspect, the polypeptide retains a phytase activity underconditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5, pH 4.0,pH 3.5, pH 3.0 or less (more acidic) pH. Alternatively, a polypeptide ofthe invention can retain a phytase activity under conditions comprisingabout pH 7.5, pH 8, pH 8.5, pH 9, pH 9.5, pH 10.0, pH 10.5, pH 11.0, pH11.5, pH 12, pH 12.5 or more (more basic) pH.

The invention provides protein preparations comprising a polypeptide ofthe invention, wherein the protein preparation comprises a liquid, aslurry, a powder, a spray, a suspension, a lyophilizedcomposition/formulation, a solid, geltab, pill, implant, a gel; or apharmaceutical formulation, a food, a feed, a food supplement, a feedsupplement, a food additive, a feed additive, a nutritional supplementor a dietary supplement thereof.

The invention provides heterodimers comprising a polypeptide of theinvention, and in one aspect the heterodimer comprises a second domain,wherein optionally the second domain is a polypeptide and theheterodimer is a fusion protein, and optionally the second domain is anepitope or a tag.

The invention provides immobilized polypeptides comprising a polypeptideof the invention, wherein the immobilized polypeptide can comprises ahomodimer or a heterodimer of the invention, wherein optionally thepolypeptide is immobilized on or inside a cell, a vesicle, a liposome, afilm, a membrane, a metal, a resin, a polymer, a ceramic, a glass, amicroelectrode, a graphitic particle, a bead, a gel, a plate, an array,a capillary tube, a crystal, a tablet, a pill, a capsule, a powder, anagglomerate, a surface, or a porous structure. In one aspect, theinvention provides arrays (e.g., microarrays) comprising an immobilizedpolypeptide, wherein the polypeptide comprises the polypeptide of theinvention, or the heterodimer of the invention, or the nucleic acid ofthe invention, or a combination thereof.

The invention provides isolated, synthetic or recombinant antibodiesthat specifically bind to a polypeptide of the invention or to apolypeptide encoded by the nucleic acid of the invention, whereinoptionally the antibody is a monoclonal or a polyclonal antibody. Theinvention provides hybridomas comprising the antibody of the invention.

The invention provides methods of producing a recombinant polypeptidecomprising: (a) providing a nucleic acid, wherein the nucleic acidcomprises a sequence of the invention; and (b) expressing the nucleicacid of (a) under conditions that allow expression of the polypeptide,thereby producing a recombinant polypeptide, and optionally the methodfurther comprises transforming a host cell with the nucleic acid of (a)followed by expressing the nucleic acid of (a), thereby producing therecombinant polypeptide in a transformed host cell. In an alternativeembodiment, the nucleic acid is operably linked to a promoter beforebeing transformed into a host cell.

The invention provides methods for identifying a polypeptide having aphytase activity comprising: (a) providing the polypeptide of theinvention; (b) providing a phytase substrate; and (c) contacting thepolypeptide or a fragment or variant thereof of (a) with the substrateof (b) and detecting an increase in the amount of substrate or adecrease in the amount of reaction product, wherein a decrease in theamount of the substrate or an increase in the amount of the reactionproduct detects a polypeptide having a phytase activity.

The invention provides methods for identifying a phytase substratecomprising: (a) providing the polypeptide of the invention; (b)providing a test substrate; and (c) contacting the polypeptide of (a)with the test substrate of (b) and detecting an increase in the amountof substrate or a decrease in the amount of reaction product, wherein adecrease in the amount of the substrate or an increase in the amount ofthe reaction product identifies the test substrate as a phytasesubstrate.

The invention provides methods of determining whether a compoundspecifically binds to a polypeptide comprising: (a) expressing a nucleicacid or a vector comprising the nucleic acid under conditions permissivefor translation of the nucleic acid to a polypeptide, wherein thenucleic acid comprises a sequence of the invention; (b) contacting thepolypeptide with the test compound; and (c) determining whether the testcompound specifically binds to the polypeptide, thereby determining thatthe compound specifically binds to the polypeptide.

The invention provides methods for identifying a modulator of a phytaseactivity comprising: (a) providing the phytase polypeptide of theinvention; (b) providing a test compound; (c) contacting the polypeptideof (a) with the test compound of (b) and measuring an activity of thephytase, wherein a change in the phytase activity measured in thepresence of the test compound compared to the activity in the absence ofthe test compound provides a determination that the test compoundmodulates the phytase activity, wherein optionally the phytase activityis measured by providing a phytase substrate and detecting an increasein the amount of the substrate or a decrease in the amount of a reactionproduct, and optionally a decrease in the amount of the substrate or anincrease in the amount of the reaction product with the test compound ascompared to the amount of substrate or reaction product without the testcompound identifies the test compound as an activator of phytaseactivity, and optionally an increase in the amount of the substrate or adecrease in the amount of the reaction product with the test compound ascompared to the amount of substrate or reaction product without the testcompound identifies the test compound as an inhibitor of phytaseactivity.

The invention provides methods for hydrolyzing an inositol-hexaphosphateto inositol and inorganic phosphate comprising: (a) providing apolypeptide having a phytase activity, wherein the polypeptide comprisesthe amino acid sequence of the invention, or, a polypeptide encoded bythe nucleic acid of the invention; (b) providing a compositioncomprising an inositol-hexaphosphate; and (c) contacting the polypeptideof (a) with the composition of (b) under conditions wherein thepolypeptide hydrolyzes the inositol-hexaphosphate to produce to inositoland inorganic phosphate, wherein optionally the conditions comprise atemperature of between about 37° C. and about 70° C., between about 50°C. and about 80° C., or between about 60° C. and about 90° C., andoptionally the composition comprises a phytic acid.

The invention provides methods for oil degumming comprising: (a)providing a polypeptide having a phytase activity, wherein thepolypeptide comprises the amino acid sequence of the invention, or, apolypeptide encoded by the nucleic acid of the invention; (b) providinga composition comprising a vegetable oil; and (c) contacting thepolypeptide of (a) and the vegetable oil of (b) under conditions whereinthe polypeptide can cleave an inositol-inorganic phosphate linkage,thereby degumming the vegetable oil.

The invention provides methods for producing a feed or a food, or a feedor food supplement, or a food or feed additive, or a nutritionalsupplement, or a dietary supplement, comprising: (a) transforming aplant, plant part or plant cell with a polynucleotide encoding a phytaseenzyme polypeptide, wherein the phytase comprises a polypeptidecomprising the amino acid sequence of the invention, or, a polypeptideencoded by the nucleic acid of the invention; (b) culturing the plant,plant part or plant cell under conditions in which the phytase enzyme isexpressed; and, (c) converting the plant, plant parts or plant cell intoa composition suitable for a food, a feed, a food supplement, a feedsupplement, a food additive, a feed additive, a nutritional supplementor a dietary supplement, or adding the cultured plant, plant part orplant cell to a food, a feed, a food supplement, a feed supplement, afood additive, a feed additive, a nutritional supplement or a dietarysupplement, thereby producing a food, a feed, a food supplement, a feedsupplement, a food additive, a feed additive, a nutritional supplementor a dietary supplement, wherein optionally the polynucleotide iscontained in an expression vector, and optionally the vector comprisesan expression control sequence capable of expressing the nucleic acid ina plant cell, and optionally the food, feed, food supplement, feedsupplement, food additive, feed additive, nutritional supplement ordietary supplement is for an animal, and optionally wherein the animalis a monogastric animal, and optionally the animal is a ruminant, andoptionally the food, feed, food supplement, feed supplement, foodadditive, feed additive, nutritional supplement or dietary supplement,is in the form of a delivery matrix, a pellet, a tablet, a gel, aliquids, a spray, ground grain or a powder. In one aspect, the phytaseenzyme is glycosylated to provide thermotolerance or thermostability atpelletizing conditions, and optionally delivery matrix is formed bypelletizing a mixture comprising a grain germ and the phytase enzyme toyield a particle, and optionally the pellets are made under conditionscomprising application of steam, optionally the pellets are made underconditions comprising application of a temperature in excess of 80° C.for about 5 minutes, and optionally the pellet comprises a phytaseenzyme that comprises a specific activity of at least 350 to about 900units per milligram of enzyme.

The invention provides methods for delivering a phytase enzymesupplement to an animal or a human, said method comprising: (a)preparing an edible delivery matrix comprising an edible carrier and aphytase enzyme comprising a polypeptide comprising the amino acidsequence of the invention, wherein the matrix readily disperses andreleases the phytase enzyme when placed into aqueous media, and, (b)administering the edible enzyme delivery matrix to the animal or human,wherein optionally in the edible delivery matrix comprises a granulateedible carrier, and optionally the edible delivery matrix is in the formof pellets, tablets, gels, liquids, sprays or powders, and optionallythe edible carrier comprises a carrier selected from the groupconsisting of grain germ, hay, alfalfa, timothy, soy hull, sunflowerseed meal, corn meal, soy meal and wheat meal, and optionally the ediblecarrier comprises grain germ that is spent of oil.

The invention provides a food, feed, food supplement, feed supplement,food additive, feed additive, nutritional supplement or dietarysupplement, for an animal or a human, comprising the polypeptide of theinvention, or a homodimer or heterodimer of the invention; whereinoptionally the polypeptide is glycosylated, and optionally the phytaseactivity is thermotolerant or thermostable. In one aspect, the food,feed, food supplement, feed supplement, food additive, feed additive,nutritional supplement or dietary supplement is manufactured in pellet,pill, tablet, capsule, gel, geltab, spray, powder, lyophilizedformulation, pharmaceutical formulation, liquid form, as a suspension orslurry, or produced using polymer-coated additives, or manufactured ingranulate form, or produced by spray drying.

The invention provides edible or absorbable enzyme delivery matrix(matrices) comprising the polypeptide of the invention, or a homodimeror heterodimer of the invention; wherein optionally the polypeptide isglycosylated, and optionally the phytase activity is thermotolerant orthermostable. In one aspect, the edible delivery matrix comprises apellet, or the edible or absorbable enzyme delivery matrix ismanufactured in pellet, pill, tablet, capsule, gel, geltab, spray,powder, lyophilized formulation, pharmaceutical formulation, liquidform, as a suspension or slurry, or produced using polymer-coatedadditives, or manufactured in granulate form, or produced by spraydrying.

The invention provides edible or absorbable pellets comprising agranulate edible or absorbable carrier and the polypeptide of theinvention, or a homodimer or heterodimer of the invention; whereinoptionally the polypeptide is glycosylated, and optionally the phytaseactivity is thermotolerant or thermostable, and optionally the pellet ismanufactured in pellet form, or as a pill, tablet, capsule, gel, geltab,spray, powder, lyophilized formulation, pharmaceutical formulation,liquid form, as a suspension or slurry, or produced using polymer-coatedadditives, or manufactured in granulate form, or produced by spraydrying.

The invention provides meals, e.g., a soybean meal, comprising apolypeptide of the invention, or a homodimer or heterodimer of theinvention, and optionally the meal, e.g., soybean meal, is manufacturedas a pellet, pill, tablet, capsule, gel, geltab, spray, powder,lyophilized formulation, or liquid form.

The invention provides methods of increasing the resistance of a phytasepolypeptide to enzymatic inactivation in a digestive system of ananimal, the method comprising glycosylating a phytase polypeptidecomprising the polypeptide of the invention, thereby increasingresistance of the phytase polypeptide to enzymatic inactivation in adigestive system of an animal, and optionally the glycosylation isN-linked glycosylation, and, and optionally the phytase polypeptide isglycosylated as a result of in vivo expression of a polynucleotideencoding the phytase in a cell, and optionally the cell is a eukaryoticcell, and optionally the eukaryotic cell is a fungal cell, a plant cell,or a mammalian cell.

The invention provides methods for processing of corn and sorghumkernels comprising: (a) providing a polypeptide having a phytaseactivity, wherein the polypeptide comprises the polypeptide of theinvention; (b) providing a composition comprising a corn steep liquor ora sorghum steep liquor; and (c) contacting the polypeptide of (a) andthe composition of (b) under conditions wherein the polypeptide cancleave an inositol-inorganic phosphate linkage.

The invention provides pharmaceuticals or a dietary formulationscomprising a polypeptide or heterodimer of the invention; whereinoptionally the polypeptide is glycosylated, and optionally the phytaseactivity is thermotolerant or thermostable; and optionally thepharmaceutical or a dietary formulation is formulated as in pellet,pill, tablet, capsule, geltab, spray, powder, lotion or liquid form, orproduced using polymer-coated additives, or as an implant, ormanufactured in granulate form, or produced by spray drying.

The invention provides compositions comprising a polypeptide orheterodimer of the invention; and, (b) any product as set forth in Table2, or any of the compositions listed in Table 1; wherein optionally thepolypeptide is glycosylated, and optionally the phytase activity isthermotolerant or thermostable.

The invention provides a self-contained meal Ready-to-Eat unit (MRE), adrink or a hydrating agent comprising a polypeptide or heterodimer ofthe invention; wherein optionally the polypeptide is glycosylated, andoptionally the phytase activity is thermotolerant or thermostable.

The invention provides methods for ameliorating (slowing the progressof, treating or preventing) osteoporosis comprising administering to anindividual in need thereof an effective amount (dosage) of a compositioncomprising a polypeptide or heterodimer of the invention; whereinoptionally the polypeptide is glycosylated, and optionally the phytaseactivity is thermotolerant or thermostable.

The invention provides methods for increasing gastric lability of aphytase comprising providing a polypeptide having a phytase activity,and replacing one or more amino acids in the sequence encoding thepolypeptide with arginine, histidine, proline, leucine, serine,threonine, or tyrosine. The invention further provides exemplaryphytases for use in the method, e.g. SEQ ID NO:2 or other phytases ofthe invention, as described in detail in Example 2, below.

The invention provides methods for altering at least two differentproperties of an enzyme, as described in detail in Example 2, below,comprising (a) providing a polypeptide having an enzymatic activity; (b)creating variants from the polypeptide of (a), wherein each variant hasa single amino acid change from the polypeptide of (a); (c) screeningthe variants of (b) for the two different properties; (d) selectingdesired variants of (c), and identifying the single amino acid change ineach selected variant; (e) creating new variants comprising differentcombinations of the selected single amino acid changes of (d); (f)screening the variants of (e) for the two altered properties; and (g)selecting desired variants of (f) with the two altered properties. Theinvention provides alternative methods for altering at least twodifferent properties of an enzyme comprising (a) providing a polypeptidehaving an enzymatic activity; (b) creating variants from the polypeptideof (a), wherein each variant has a single amino acid change from thepolypeptide of (a); (c) screening the variants of (b) for one alteredproperty; (d) screening the variants (b) for another altered property;(e) selecting desired variants of (c) and (d), and identifying thesingle amino acid change in each selected variant; (f) creating newvariants comprising different combinations of the selected single aminoacid changes of (c) and (d); (g) screening the variants of (f) for thetwo altered properties; and (h) selecting desired variants of (g) withthe two altered properties. The invention further provides exemplarymethods for creating the variants, e.g. by GSSM evolution,GeneReassembly evolution, and/or TMCA evolution.

The invention provides methods for decoupling gastric stability fromthermotolerance of a phytase, as described in detail in Example 2,below, comprising (a) providing a polypeptide having a phytase activity;(b) creating variant phytases from the polypeptide of (a), wherein eachvariant has a single amino acid change from the polypeptide of (a); (c)screening the variant phytases of (b) for altered gastric stability andaltered thermotolerance; (d) selecting the variants of (c) with desiredgastric stability and thermotolerance, and identifying the single aminoacid change in each selected variant; (e) creating new variant phytasescomprising different combinations of the selected single amino acidchanges of (d); (f) screening the variants of (e) for altered gastricstability and altered thermotolerance; and (g) selecting the variants of(f) with desired gastric stability and thermotolerance. The inventionprovides alternative methods for decoupling gastric stability fromthermotolerance of a phytase comprising (a) providing a polypeptidehaving a phytase activity; (b) creating variant phytases from thepolypeptide of (a), wherein each variant has a single amino acid changefrom the polypeptide of (a); (c) screening the variant phytases of (b)for altered gastric stability; (d) screening the variant phytases (b)for altered thermotolerance; (e) selecting the variants of (c) and (d)with desired gastric stability or thermotolerance, and identifying thesingle amino acid change in each selected variant; (f) creating newvariant phytases comprising different combinations of the selectedsingle amino acid changes of (c) and (d); (g) screening the variants of(f) for altered gastric stability and altered thermotolerance; and (h)selecting the variants of (g) with desired gastric stability andthermotolerance. The invention further provides exemplary methods forcreating the variants, e.g. by GSSM evolution, GeneReassembly evolution,and/or TMCA evolution. The invention provides exemplary phytases for usein the methods, e.g. SEQ ID NO:2 and other phytases of the invention.The invention provides exemplary mutations which decouple gastricstability from thermotolerance, for example, the mutations listed inTable 4, 5, 6, 7, 9, or any combination thereof.

The invention provides phytases which are gastric labile andthermotolerant, described in detail in Example 2, below, wherein thephytase completely degrades in stimulated gastric fluid (SGF) in lessthan 10 minutes, less than 8 minutes, less than 6 minutes, less than 4minutes, or less than 2 minutes, and the phytase retains activity afterexposure to a temperature in the range from about 75° C. to about 85°C., about 85° C. to about 90° C., about 90° C. to about 95° C., or about80° C. to about 86° C., for example, the phytases of the invention asdescribed in detail in Example 2, below. In one aspect, the gastriclabile and thermotolerant phytases of the invention completely degradein stimulated gastric fluid (SGF) in less than 4 minutes, and thephytase retains activity after exposure to a temperature in the rangefrom about 80° C. to about 86° C.

The details of one or more aspects of the invention are set forth in theaccompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of aspects of the invention andare not meant to limit the scope of the invention as encompassed by theclaims.

FIG. 1 illustrates a summary of residue activity of purifiedpolypeptides of the invention, single mutation exemplary sequences ofthe invention, after heat treatment at various temperatures for 30minutes; where the phytase activity is assayed with a fluorescencesubstrate, and the rates were compared to the rates of eachcorresponding non-treated sample; as described in detail in Example 1,below.

FIG. 2 illustrates a summary of residue activity of purifiedpolypeptides of the invention comprising “blended” single residuemutations (phytases containing multiple mutations), after heat treatmenton a thermocycler; where the phytase activity is assayed with afluorescence substrate, and the rates were compared to the rates of eachcorresponding non-treated sample; as summarized in FIG. 1; as describedin detail in Example 1, below.

FIG. 3 graphically summarizes the data used to generate the graph ofFIG. 1; as described in detail in Example 1, below.

FIG. 4 illustrates the sequence of the parental phytase SEQ ID NO:2 andthe gene site saturation mutagenesis (GSSM)-generated sequencemodifications selected for GeneReassembly™ library construction; asdescribed in detail, below.

FIG. 5 illustrates exemplary phytases having multiple residuemodifications to the parental SEQ ID NO:2; as described in detailherein.

FIG. 6 illustrates exemplary phytases having single residuemodifications to the parental SEQ ID NO:2; as described in detailherein.

FIG. 7 schematically illustrates an exemplary phytase assay of theinvention using the fluorescence substrate 4-methylumbelliferylphosphate (MeUMB-phosphate); as described in detail in Example 1, below.

FIG. 8 schematically illustrates an exemplary phytase assay of theinvention that uses the fluorescence substrate MeUMB-phosphate; asdescribed in detail in Example 1, below.

FIG. 9 schematically illustrates the protocol for an exemplary libraryscreen, as described in FIG. 8, as described in detail in Example 1,below.

FIG. 10 illustrates an exemplary alcohol process that can incorporateuse of phytases of this invention.

FIGS. 11A and 11B illustrate summaries of thermostability and SGFlability of appA phytase (GenBank accession no. M58708), appA-SEQ IDNO:2 intermediates, and SEQ ID NO:2, as described in detail in Example2, below.

FIG. 12 illustrates the effect of a his-tag on SGF stability, asdetermined by SGF assays of purified his-tag and non his-tag versions ofthe parental phytase (SEQ ID NO:2), as described in detail in Example 2,below.

FIG. 13 illustrates the thermotolerance of glycosylation-minus SEQ IDNO:2 variants (Variants GLY1-GLY4) and two SEQ ID NO:2 controls, asdescribed in detail in Example 2, below.

FIG. 14 illustrates the SGF stability of SEQ ID NO:2-HIS and the SEQ IDNO:2-6X variant, as described in detail in Example 2, below.

FIG. 15 illustrates the SGF activity loss of select mutants from GSSMevolution of SEQ ID NO:2, as described in detail in Example 2, below.

FIG. 16 illustrates the effect of different amino acids on SGF lability,as described in detail in Example 2, below.

FIG. 17 illustrates SGF mutation hot spots, as described in detail inExample 2, below.

FIG. 18 illustrates SGF lability of SEQ ID NO:2-HIS and Variant O atdifferent pepsin dosages, as described in detail in Example 2, below.

FIG. 19 illustrates the ½ life of SEQ ID NO:2-HIS and Variant O, asdescribed in detail in Example 2, below.

FIG. 20 illustrates the residual activity of SGF labile phytasevariants, as described in detail in Example 2, below.

FIG. 21 illustrates the specific activity of SGF label variant phytasesas compared to SEQ ID NO:2, as described in detail in Example 2, below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to phytase polypeptides having comprising thespecific residue modifications to SEQ ID NO:2, as described above, andpolynucleotides encoding them, (e.g., comprising the specific base pairmodifications to SEQ ID NO:1, as described above), as well as methods ofuse of the polynucleotides and polypeptides. FIG. 6 illustratesexemplary phytases having single residue modifications to the parentalSEQ ID NO:2, and FIG. 5 illustrates exemplary phytases having multipleresidue modifications to the parental SEQ ID NO:2.

The phytase activity of polypeptides of the invention can encompassenzymes having any phytase activity, for example, enzymes capable ofcatalyzing the degradation of phytate, e.g., the catalysis of phytate(myo-inositol-hexaphosphate) to inositol and inorganic phosphate. Thephytases of the invention include thermotolerant and thermoresistantenzymes.

The phytases and polynucleotides encoding the phytases of the inventionare useful in a number of processes, methods, and compositions. Forexample, as discussed above, a phytase can be used in animal feed, andfeed supplements as well as in treatments to degrade or remove excessphytate from the environment or a sample. Other uses will be apparent tothose of skill in the art based upon the teachings provided herein,including those discussed above.

In one aspect, phytase molecules of the invention—either alone or incombination with other reagents (including but not limited to enzymes,such as proteases, amylases and the like)—are used in the processing offoodstuffs, e.g., for prevention of the unwanted corn sludge, and inother applications where phytate hydrolysis is desirable.

In one aspect, phytase molecules of the invention are used to eliminateor decrease the presence of unhydrolyzed phytate, especially whereunhydrolyzed phytate leads to problematic consequences in ex vivoprocesses including—but not limited to—the processing of foodstuffs. Inone aspect, phytase molecules of the invention are used in procedures asdescribed in EP0321004-B1 (Vaara et al.), including steps in theprocessing of corn and sorghum kernels whereby the hard kernels aresteeped in water to soften them. Water-soluble substances that leach outduring this process become part of a corn steep liquor, which isconcentrated by evaporation. Unhydrolyzed phytic acid in the corn steepliquor, largely in the form of calcium and magnesium salts, isassociated with phosphorus and deposits an undesirable sludge withproteins and metal ions. This sludge is problematic in the evaporation,transportation and storage of the corn steep liquor. Phytase moleculesof the invention are used to hydrolyze this sludge.

In one aspect, the phytases of the invention can provide substantiallysuperior commercial performance than previously identified phytasemolecules, e.g. phytase molecules of fungal origin. In one aspect, theenzymes of the invention can be approximately 4400 U/mg, or greater thanapproximately between 50 to 100, or 50 to 1000, or 100 to 4000 U/mgprotein.

The invention also provides methods for changing the characteristics ofa phytase of the invention by mutagenesis and other method, as discussedin detail, below.

Generating and Manipulating Nucleic Acids

The invention provides nucleic acids encoding the polypeptides andphytases of the invention. The invention also provides expressioncassettes, vectors such as expression or cloning vectors, cloningvehicles such as a viral vector, a plasmid, a phage, a phagemid, acosmid, a fosmid, a bacteriophage or an artificial chromosome, which cancomprise, or have contained therein, a nucleic acid of the invention.

The invention also includes methods for discovering new phytasesequences using the nucleic acids of the invention. Also provided aremethods for modifying the nucleic acids of the invention by, e.g.,synthetic ligation reassembly, optimized directed evolution systemand/or saturation mutagenesis.

In one aspect, the invention provides a genus of nucleic acids that aresynthetically generated variants of the parent SEQ ID NO:1, whereinthese nucleic acids of the invention having at least 95%, 96% 97%, 98%or 99% sequence identity to the “parent” SEQ ID NO:1, and encoding atleast one mutation listed in Table 4, 5, 6, 7, 9, or any combinationthereof,

For reference, the parent SEQ ID NO:1 is:

atgaaagcga tcttaatccc atttttatct cttctgattc cgttaacccc gcaatctgca   60ttcgctcaga gtgagccgga gctgaagctg gaaagtgtgg tgattgtcag tcgtcatggt  120gtgcgtgctc caaccaaggc cacgcaactg atgcaggatg tcaccccaga cgcatggcca  180acctggccgg taaaactggg tgagctgaca ccgcgcggtg gtgagctaat cgcctatctc  240ggacattact ggcgtcagcg tctggtagcc gacggattgc tgcctaaatg tggctgcccg  300cagtctggtc aggtcgcgat tattgctgat gtcgacgagc gtacccgtaa aacaggcgaa  360gccttcgccg ccgggctggc acctgactgt gcaataaccg tacataccca ggcagatacg  420tccagtcccg atccgttatt taatcctcta aaaactggcg tttgccaact ggataacgcg  480aacgtgactg acgcgatcct cgagagggca ggagggtcaa ttgctgactt taccgggcat  540tatcaaacgg cgtttcgcga actggaacgg gtgcttaatt ttccgcaatc aaacttgtgc  600cttaaacgtg agaaacagga cgaaagctgt tcattaacgc aggcattacc atcggaactc  660aaggtgagcg ccgactgtgt ctcattaacc ggtgcggtaa gcctcgcatc aatgctgacg  720gagatatttc tcctgcaaca agcacaggga atgccggagc cggggtgggg aaggatcacc  780gattcacacc agtggaacac cttgctaagt ttgcataacg cgcaatttga tttgctacaa  840cgcacgccag aggttgcccg cagccgcgcc accccgttat tagatttgat caagacagcg  900ttgacgcccc atccaccgca aaaacaggcg tatggtgtga cattacccac ttcagtgctg  960tttatcgccg gacacgatac taatctggca aatctcggcg gcgcactgga gctcaactgg 1020acgcttcccg gtcagccgga taacacgccg ccaggtggtg aactggtgtt tgaacgctgg 1080cgtcggctaa gcgataacag ccagtggatt caggtttcgc tggtcttcca gactttacag 1140cagatgcgtg ataaaacgcc gctgtcatta aatacgccgc ccggagaggt gaaactgacc 1200ctggcaggat gtgaagagcg aaatgcgcag ggcatgtgtt cgttggcagg ttttacgcaa 1260atcgtgaatg aagcacgcat accggcgtgc agtttgtaa 1299

The nucleic acids of the invention can be made, isolated and/ormanipulated by, e.g., cloning and expression of cDNA libraries,amplification of message or genomic DNA by PCR, and the like. Inpracticing the methods of the invention, homologous genes can bemodified by manipulating a template nucleic acid, as described herein.The invention can be practiced in conjunction with any method orprotocol or device known in the art, which are well described in thescientific and patent literature.

General Techniques

The nucleic acids used to practice this invention, whether RNA, iRNA,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides generated from these nucleic acids can beindividually isolated or cloned and tested for a desired activity. Anyrecombinant expression system can be used, including bacterial,mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

In alternative aspects, the phrases “nucleic acid” or “nucleic acidsequence” refer to an oligonucleotide, nucleotide, polynucleotide, or toa fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA) ofgenomic or synthetic origin which may be single-stranded ordouble-stranded and may represent a sense or antisense strand, topeptide nucleic acid (PNA), or to any DNA-like or RNA-like material,natural or synthetic in origin, including, e.g., iRNA,ribonucleoproteins (e.g., iRNPs). The term encompasses nucleic acids,i.e., oligonucleotides, containing known analogues of naturalnucleotides. The term also encompasses nucleic-acid-like structures withsynthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol.144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag(1996) Antisense Nucleic Acid Drug Dev 6:153-156.

In one aspect, recombinant polynucleotides of the invention comprisesequences adjacent to a “backbone” nucleic acid to which it is notadjacent in its natural environment. In one aspect, nucleic acidsrepresent 5% or more of the number of nucleic acid inserts in apopulation of nucleic acid “backbone molecules.” “Backbone molecules”according to the invention include nucleic acids such as expressionvectors, self-replicating nucleic acids, viruses, integrating nucleicacids, and other vectors or nucleic acids used to maintain or manipulatea nucleic acid insert of interest. In one aspect, the enriched nucleicacids represent 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more ofthe number of nucleic acid inserts in the population of recombinantbackbone molecules.

In one aspect, a nucleic acid encoding a polypeptide of the invention isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide of the invention can be fused to a heterologous peptide orpolypeptide, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification. Peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate antibodies and antibody-expressing B cells, and the like.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

In one aspect, the term “isolated” means that the material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.In one aspect, the term “purified” does not require absolute purity;rather, it is intended as a relative definition. Individual nucleicacids obtained from a library have been conventionally purified toelectrophoretic homogeneity. The sequences obtained from these clonescould not be obtained directly either from the library or from totalhuman DNA. The purified nucleic acids of the invention have beenpurified from the remainder of the genomic DNA in the organism by atleast 10⁴-10⁶ fold. In alternative aspects, the term “purified” alsoincludes nucleic acids which have been purified from the remainder ofthe genomic DNA or from other sequences in a library or otherenvironment by at least one order of magnitude, or alternatively, two orthree orders, or four or five orders of magnitude.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA) sequences of theinvention operatively linked to expression (e.g., transcriptional ortranslational) control sequence(s), e.g., promoters or enhancers, todirect or modulate RNA synthesis/expression. The expression controlsequence can be in an expression vector. Exemplary bacterial promotersinclude lad, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplaryeukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein I.

Promoters suitable for expressing, or over-expressing, a polypeptide inbacteria include the E. coli lac or trp promoters, the lacI promoter,the lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter,the lambda PR promoter, the lambda PL promoter, promoters from operonsencoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), andthe acid phosphatase promoter. Eukaryotic promoters include the CMVimmediate early promoter, the HSV thymidine kinase promoter, heat shockpromoters, the early and late SV40 promoter, LTRs from retroviruses, andthe mouse metallothionein-I promoter. Other promoters known to controlexpression of genes in prokaryotic or eukaryotic cells or their virusesmay also be used.

Expression Vectors and Cloning Vehicles

The invention provides expression systems, e.g., expression cassettes,vectors, cloning vehicles and the like, comprising nucleic acids of theinvention, e.g., sequences encoding the phytases of the invention, forexpression, and over-expression, of the polypeptides of the invention(and nucleic acids, e.g., antisense). Expression vectors and cloningvehicles of the invention can comprise viral particles, baculovirus,phage, plasmids, phagemids, cosmids, fosmids, bacterial artificialchromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox virus,pseudorabies and derivatives of SV40), P1-based artificial chromosomes,yeast plasmids, yeast artificial chromosomes, and any other vectorsspecific for specific hosts of interest (such as bacillus, Aspergillusand yeast). Vectors of the invention can include chromosomal,non-chromosomal and synthetic DNA sequences. Large numbers of suitablevectors are known to those of skill in the art, and are commerciallyavailable. Exemplary vectors are include: bacterial: pQE vectors(Qiagen), pBluescript plasmids, pNH vectors, (lambda-ZAP vectors(Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic:pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia).However, any other plasmid or other vector may be used so long as theyare replicable and viable in the host. Low copy number or high copynumber vectors may be employed with the present invention.

As representative examples of expression vectors which may be used theremay be mentioned viral particles, baculovirus, phage, plasmids,phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA(e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as bacillus, Aspergillus and yeast).Thus, for example, the DNA may be included in any one of a variety ofexpression vectors for expressing a polypeptide. Such vectors includechromosomal, nonchromosomal and synthetic DNA sequences. Large numbersof suitable vectors are known to those of skill in the art, and arecommercially available. The following vectors are provided by way ofexample; Bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNHvectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540,pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene), pSVK3, pBPV,pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vectormay be used so long as they are replicable and viable in the host. Lowcopy number or high copy number vectors may be employed with the presentinvention.

An exemplary vector for use in the present invention contains anf-factor origin replication. The f-factor (or fertility factor) in E.coli is a plasmid which effects high frequency transfer of itself duringconjugation and less frequent transfer of the bacterial chromosomeitself. One aspect uses cloning vectors, referred to as “fosmids” orbacterial artificial chromosome (BAC) vectors. These are derived from E.coli f-factor which is able to stably integrate large segments ofgenomic DNA. When integrated with DNA from a mixed unculturedenvironmental sample, this makes it possible to achieve large genomicfragments in the form of a stable “environmental DNA library.”

Another type of vector for use in the present invention is a cosmidvector. Cosmid vectors were originally designed to clone and propagatelarge segments of genomic DNA. Cloning into cosmid vectors is describedin detail in “Molecular Cloning: A laboratory Manual” (Sambrook et al.,1989).

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct RNAsynthesis. Particular named bacterial promoters include lad, lacZ, T3,T7, gpt, lambda P_(R), P_(L) and trp. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus, and mouse metallothionein-I. Selection of the appropriatevector and promoter is well within the level of ordinary skill in theart. The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Promoterregions can be selected from any desired gene using CAT (chloramphenicoltransferase) vectors or other vectors with selectable markers. Inaddition, the expression vectors can contain one or more selectablemarker genes to provide a phenotypic trait for selection of transformedhost cells such as dihydrofolate reductase or neomycin resistance foreukaryotic cell culture, or tetracycline or ampicillin resistance in E.coli.

In one aspect, expression cassettes of the invention comprise a sequenceof the invention and a nucleotide sequence which is capable of affectingexpression of a structural gene (i.e., a protein coding sequence, suchas a phytase of the invention) in a host compatible with such sequences.Expression cassettes include at least a promoter operably linked withthe polypeptide coding sequence; and, optionally, with other sequences,e.g., transcription termination signals. Additional factors necessary orhelpful in effecting expression may also be used, e.g., enhancers. Inone aspect, “operably linked” as used herein refers to linkage of apromoter upstream from a DNA sequence such that the promoter mediatestranscription of the DNA sequence. Thus, expression cassettes alsoinclude plasmids, expression vectors, recombinant viruses, any form ofrecombinant “naked DNA” vector, and the like. In one aspect, a “vector”comprises a nucleic acid that can infect, transfect, transiently orpermanently transduce a cell. A vector can be a naked nucleic acid, or anucleic acid complexed with protein or lipid. The vector optionallycomprises viral or bacterial nucleic acids and/or proteins, and/ormembranes (e.g., a cell membrane, a viral lipid envelope, etc.). In oneaspect, vectors include, but are not limited to, replicons (e.g., RNAreplicons, bacteriophages) to which fragments of DNA may be attached andbecome replicated. In one aspect, vectors include, but are not limitedto RNA, autonomous self-replicating circular or linear DNA or RNA (e.g.,plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879),and includes both the expression and non-expression plasmids. Where arecombinant microorganism or cell culture is described as hosting an“expression vector” this includes both extra-chromosomal circular andlinear DNA and DNA that has been incorporated into the hostchromosome(s). Where a vector is being maintained by a host cell, thevector may either be stably replicated by the cells during mitosis as anautonomous structure, or is incorporated within the host's genome.

The expression vector may comprise a promoter, a ribosome binding sitefor translation initiation and a transcription terminator. The vectormay also include appropriate sequences for amplifying expression.Mammalian expression vectors can comprise an origin of replication, anynecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

In one aspect, the expression vectors contain one or more selectablemarker genes to permit selection of host cells containing the vector.Such selectable markers include genes encoding dihydrofolate reductaseor genes conferring neomycin resistance for eukaryotic cell culture,genes conferring tetracycline or ampicillin resistance in E. coli, andthe S. cerevisiae TRP1 gene. Promoter regions can be selected from anydesired gene using chloramphenicol transferase (CAT) vectors or othervectors with selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells may also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin bp 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

A DNA sequence may be inserted into a vector by a variety of procedures.In general, the DNA sequence is ligated to the desired position in thevector following digestion of the insert and the vector with appropriaterestriction endonucleases. Alternatively, blunt ends in both the insertand the vector may be ligated. A variety of cloning techniques are knownin the art, e.g., as described in Ausubel and Sambrook. Such proceduresand others are deemed to be within the scope of those skilled in theart.

The vector may be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook.

Particular bacterial vectors which may be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

Host Cells and Transformed Cells

The invention also provides a transformed cell comprising a nucleic acidsequence of the invention, e.g., a sequence encoding a phytase of theinvention, or comprising an expression cassette, vector, cloningvehicle, expression vector, or cloning vector of the invention. The hostcell may be any of the host cells familiar to those skilled in the art,including prokaryotic cells, eukaryotic cells, such as bacterial cells,fungal cells, yeast cells, mammalian cells, insect cells, or plantcells. Exemplary bacterial cells include any species within the generaEscherichia, Bacillus, Streptomyces, Salmonella, Pseudomonas,Lactococcus, and Staphylococcus, including, e.g., Escherichia coli,Lactococcus lactic, Bacillus subtilis, Bacillus cereus, Salmonellatyphimurium, Pseudomonas fluorescens. Exemplary fungal cells include anyspecies of Aspergillus, including Aspergillus niger. Exemplary yeastcells include any species of Pichia, Saccharomyces, Schizosaccharomyces,or Schwanniomyces, including Pichia pastoris, Saccharomyces cerevisiae,or Schizosaccharomyces pombe. Exemplary insect cells include any speciesof Spodoptera or Drosophila, including Drosophila S2 and Spodoptera S/9.Exemplary insect cells include Drosophila S2 and Spodoptera Sf9.Exemplary yeast cells include Pichia pastoris, Saccharomyces cerevisiaeor Schizosaccharomyces pombe. Exemplary animal cells include CHO, COS orBowes melanoma or any mouse or human cell line. The selection of anappropriate host is within the abilities of those skilled in the art.

The vector may be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce apolypeptide of the invention. Cell-free translation systems can usemRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

The nucleic acids of the invention can be expressed, or overexpressed,in any in vitro or in vivo expression system. Any cell culture systemscan be employed to express, or over-express, recombinant protein,including bacterial, insect, yeast, fungal or mammalian cultures.Over-expression can be effected by appropriate choice of promoters,enhancers, vectors (e.g., use of replicon vectors, dicistronic vectors(see, e.g., Gurtu (1996) Biochem. Biophys. Res. Commun. 229:295-8)),media, culture systems and the like. In one aspect, gene amplificationusing selection markers, e.g., glutamine synthetase (see, e.g., Sanders(1987) Dev. Biol. Stand. 66:55-63), in cell systems are used tooverexpress the polypeptides of the invention.

Various mammalian cell culture systems can be employed to expressrecombinant protein, examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described in“SV40-transformed simian cells support the replication of early SV40mutants” (Gluzman, 1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnon-transcribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednon-transcribed genetic elements.

Host cells containing the polynucleotides of interest can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan. The clones which are identified ashaving the specified enzyme activity may then be sequenced to identifythe polynucleotide sequence encoding an enzyme having the enhancedactivity.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids encoding the polypeptides ofthe invention, or modified nucleic acids, can be reproduced by, e.g.,amplification. The invention provides amplification primer sequencepairs for amplifying nucleic acids encoding polypeptides with a phytaseactivity, or subsequences thereof, where the primer pairs are capable ofamplifying nucleic acid sequences including the exemplary SEQ ID NO:1,and at least one of the specific sequence modifications set forth above.One of skill in the art can design amplification primer sequence pairsfor any part of or the full length of these sequences; for example:

The “parent” SEQ ID NO:1 is as shown above. Thus, an amplificationprimer sequence pair for amplifying this parent sequence, or one of theexemplary sequences of the invention having at least one of the specificsequence modifications set forth herein, can be residues 1 to 21 of SEQID NO:1 (i.e., ATGAAAGCGATCTTAATCCCA) and the complementary strand ofthe last 21 residues of SEQ ID NO:1 (i.e., the complementary strand ofTGCAGTTTGAGATCTCATCTA).

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (e.g., to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In one aspect of the invention, messageisolated from a cell or a cDNA library are amplified. The skilledartisan can select and design suitable oligonucleotide amplificationprimers. Amplification methods are also well known in the art, andinclude, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS,A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y.(1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y.,ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560;Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Determining the Degree of Sequence Identity

The invention provides an isolated, synthetic or recombinant nucleicacid comprising a nucleic acid sequence having at least 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO:1, and including at least one of thespecifically enumerated modifications to SEQ ID NO:1 discussed above. Inone aspect, the extent of sequence identity (homology) may be determinedusing any computer program and associated parameters, including thosedescribed herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with thedefault parameters.

Homologous sequences also include RNA sequences in which uridinesreplace the thymines in the nucleic acid sequences. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error.

Various sequence comparison programs identified herein are used in thisaspect of the invention. Protein and/or nucleic acid sequence identities(homologies) may be evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are not limited to, TBLASTN, BLASTP, FASTA,TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410,1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higginset al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol.Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272,1993.

Homology or identity can be measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection. For sequencecomparison, one sequence can act as a reference sequence (an exemplarysequence of the invention) to which test sequences are compared. Whenusing a sequence comparison algorithm, test and reference sequences areentered into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous residues. For example, inalternative aspects of the invention, continugous residues ranginganywhere from 20 to the full length of exemplary sequences of theinvention are compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned. Ifthe reference sequence has the requisite sequence identity to exemplarysequences of the invention, e.g., 98% sequence identity to SEQ ID NO:1,SEQ ID NO:2, and having one of the specific sequence modifications notedabove, that sequence is within the scope of the invention. Inalternative embodiments, subsequences ranging from about 20 to 600,about 50 to 200, and about 100 to 150 are compared to a referencesequence of the same number of contiguous positions after the twosequences are optimally aligned. Methods of alignment of sequence forcomparison are well-known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of person & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection. Other algorithmsfor determining homology or identity include, for example, in additionto a BLAST program (Basic Local Alignment Search Tool at the NationalCenter for Biological Information, such as BLAST, BLAST2, BLASTN andBLASTX), ALIGN, AMAS (Analysis of Multiply Aligned Sequences), AMPS(Protein Multiple Sequence Alignment), ASSET (Aligned SegmentStatistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN (BiologicalSequence Comparative Analysis Node), BLIMPS (BLocks IMProved Searcher),FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS,LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, Las Vegasalgorithm, FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245, 1990),FNAT (Forced Nucleotide Alignment Tool), Framealign, Framesearch,DYNAMIC, FILTER, FSAP (Fristensky Sequence Analysis Package), GAP(Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC (SensitiveSequence Comparison), LALIGN (Local Sequence Alignment), LCP (LocalContent Program), MACAW (Multiple Alignment Construction & AnalysisWorkbench), MAP (Multiple Alignment Program), MBLKP, MBLKN, PIMA(Pattern-Induced Multi-sequence Alignment), SAGA (Sequence Alignment byGenetic Algorithm) and WHAT-IF. Other programs and databases used topractice the invention include, but are not limited to: MacPattern(EMBL), DiscoveryBase (Molecular Applications Group), GeneMine(Molecular Applications Group), Look (Molecular Applications Group),MacLook (Molecular Applications Group), Catalyst (Molecular SimulationsInc.), Catalyst/SHAPE (Molecular Simulations Inc.), Cerius2.DBAccess(Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.),Insight II, (Molecular Simulations Inc.), Discover (MolecularSimulations Inc.), CHARMm (Molecular Simulations Inc.), Felix (MolecularSimulations Inc.), DelPhi, (Molecular Simulations Inc.), QuanteMM,(Molecular Simulations Inc.), Homology (Molecular Simulations Inc.),Modeler (Molecular Simulations Inc.), ISIS (Molecular Simulations Inc.),Quanta/Protein Design (Molecular Simulations Inc.), WebLab (MolecularSimulations Inc.), WebLab Diversity Explorer (Molecular SimulationsInc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (MolecularSimulations Inc.), the MDL Available Chemicals Directory database, theMDL Drug Data Report data base, the Comprehensive Medicinal Chemistrydatabase, Derwent's World Drug Index database, the BioByteMasterFiledatabase, the GenBank database, the GenSeq database, and the GenomeQuestdatabase. Many other programs and data bases would be apparent to one ofskill in the art given the present disclosure. Such alignment programscan also be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, through the NCBI (National Centerfor Biotechnology Information) website. Databases containing genomicinformation annotated with some functional information are maintained bydifferent organization, and are accessible via the internet.

BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practicethe invention. They are described, e.g., in Altschul (1977) Nuc. AcidsRes. 25:3389-3402; Altschul (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul (1990) supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. The BLAST algorithm also performs a statisticalanalysis of the similarity between two sequences (see, e.g., Karlin &Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873). One measure ofsimilarity provided by BLAST algorithm is the smallest sum probability(P(N)), which provides an indication of the probability by which a matchbetween two nucleotide or amino acid sequences would occur by chance.For example, a nucleic acid is considered similar to a referencessequence if the smallest sum probability in a comparison of the testnucleic acid to the reference nucleic acid is less than about 0.2, lessthan about 0.01, or less than about 0.001. In one aspect, protein andnucleic acid sequence homologies are evaluated using the Basic LocalAlignment Search Tool (“BLAST”). For example, five specific BLASTprograms can be used to perform the following task: (1) BLASTP andBLAST3 compare an amino acid query sequence against a protein sequencedatabase; (2) BLASTN compares a nucleotide query sequence against anucleotide sequence database; (3) BLASTX compares the six-frameconceptual translation products of a query nucleotide sequence (bothstrands) against a protein sequence database; (4) TBLASTN compares aquery protein sequence against a nucleotide sequence database translatedin all six reading frames (both strands); and, (5) TBLASTX compares thesix-frame translations of a nucleotide query sequence against thesix-frame translations of a nucleotide sequence database. The BLASTprograms identify homologous sequences by identifying similar segments,which are referred to herein as “high-scoring segment pairs,” between aquery amino or nucleic acid sequence and a test sequence which can beobtained from a protein or nucleic acid sequence database. High-scoringsegment pairs can be identified (i.e., aligned) by means of a scoringmatrix, many of which are known in the art. An exemplary scoring matrixused is the BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992;Henikoff and Henikoff, Proteins 17:49-61, 1993). Alternatively, the PAMor PAM250 matrices may be used (see, e.g., Schwartz and Dayhoff, eds.,1978, Matrices for Detecting Distance Relationships: Atlas of ProteinSequence and Structure, Washington: National Biomedical ResearchFoundation).

In one aspect of the invention, to determine if a nucleic acid has therequisite sequence identity to be within the scope of the invention, theNCBI BLAST 2.2.2 programs is used. default options to blastp. There areabout 38 setting options in the BLAST 2.2.2 program. In this exemplaryaspect of the invention, all default values are used except for thedefault filtering setting (i.e., all parameters set to default exceptfiltering which is set to OFF); in its place a “-F F” setting is used,which disables filtering. Use of default filtering often results inKarlin-Altschul violations due to short length of sequence.

The default values used in this exemplary aspect of the inventioninclude:

-   -   “Filter for low complexity: ON        -   >Word Size: 3        -   >Matrix: Blosum62        -   >Gap Costs: Existence: 11            -   >Extension: 1”

“Filter for low complexity: ON

Other default settings are: filter for low complexity OFF, word size of3 for protein, BLOSUM62 matrix, gap existence penalty of −11 and a gapextension penalty of −1.

In some aspects, a sequence comparison algorithm can be used forcomparing a nucleic acid sequence or amino acid sequence of theinvention to a reference sequence. For example, the sequence comparisonalgorithm may compare the nucleotide sequences or amino acid sequencesof the invention with the “parent” sequence SEQ ID NO:1 and/or SEQ IDNO:2, or to reference sequences to identify homologies or structuralmotifs. A comparison of the sequences can be performed to determine ifthe first sequence is the same as the second sequence. It is importantto note that this type of comparison is not limited to performing anexact comparison between the new sequence and the first sequence in thedatabase. Well-known methods are known to those of skill in the art forcomparing two nucleotide or protein sequences, even if they are notidentical. For example, gaps can be introduced into one sequence inorder to raise the homology level between the two tested sequences. Theparameters that control whether gaps or other features are introducedinto a sequence during comparison are normally entered by the user ofthe comparison algorithm.

Once a comparison of two sequences has been performed, a determinationis made whether the two sequences are the same. Of course, the term“same” is not limited to sequences that are absolutely identical. Thesequence comparison may indicate a sequence identity level between thesequences compared or identify structural motifs, or it may identifystructural motifs in sequences which are compared to these nucleic acidcodes and polypeptide codes. The level of sequence identity isdetermined by calculating the proportion of characters between thesequences that were the same out of the total number of sequences in thefirst sequence. Thus, if every character in a first 100 nucleotidesequence aligned with every character in a second sequence, the sequenceidentity level would be 100%.

Alternatively, the algorithm can compare a reference sequence to asequence of the invention to determine whether the sequences differ atone or more positions. The result of the comparison may indicate thelength and identity of inserted, deleted or substituted nucleotides oramino acid residues with respect to the sequence of either the referenceor the invention. In other aspects, the algorithm can be used toidentify features within a nucleic acid or polypeptide of the invention.For example, identifier feature may comprise an open reading frame(ORF), an “Initiation Codon” (e.g., the codon “ATG”), a “TAATAA Box”, ormotifs such as alpha helices, beta sheets, or functional polypeptidemotifs such as enzymatic active sites, helix-turn-helix motifs, leucinezippers, glycosylation sites, ubiquitination sites, alpha helices, betasheets, signal sequences encoding signal peptides which direct thesecretion of the encoded proteins, sequences implicated in transcriptionregulation such as homeoboxes, acidic stretches, enzymatic active sites,substrate binding sites, and enzymatic cleavage sites, as well as othermotifs known to those skilled in the art.

Inhibiting Expression of a Phytase

The invention further provides for nucleic acids complementary to (e.g.,antisense sequences to) the nucleic acid sequences of the invention,including nucleic acids comprising antisense, iRNA, ribozymes. Antisensesequences are capable of inhibiting the transport, splicing ortranscription of phytase-encoding genes. The inhibition can be effectedthrough the targeting of genomic DNA or messenger RNA. The transcriptionor function of targeted nucleic acid can be inhibited, for example, byhybridization and/or cleavage. One particularly useful set of inhibitorsprovided by the present invention includes oligonucleotides which areable to either bind phytase gene or message, in either case preventingor inhibiting the production or function of phytase enzyme. Theassociation can be though sequence specific hybridization. Anotheruseful class of inhibitors includes oligonucleotides which causeinactivation or cleavage of phytase message. The oligonucleotide canhave enzyme activity which causes such cleavage, such as ribozymes. Theoligonucleotide can be chemically modified or conjugated to an enzyme orcomposition capable of cleaving the complementary nucleic acid. One mayscreen a pool of many different such oligonucleotides for those with thedesired activity.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides comprising the newphytase sequence modifications of the invention, where these antisenseoligonucleotides are capable of binding phytase message which caninhibit phytase activity by targeting mRNA. Strategies for designingantisense oligonucleotides are well described in the scientific andpatent literature, and the skilled artisan can design such phytaseoligonucleotides using the novel reagents of the invention. For example,gene walking/RNA mapping protocols to screen for effective antisenseoligonucleotides are well known in the art, see, e.g., Ho (2000) MethodsEnzymol. 314:168-183, describing an RNA mapping assay, which is based onstandard molecular techniques to provide an easy and reliable method forpotent antisense sequence selection. See also Smith (2000) Euro. J.Pharm. Sci. 11:191-198.

Naturally occurring nucleic acids are used as antisenseoligonucleotides. The antisense oligonucleotides can be of any length;for example, in alternative aspects, the antisense oligonucleotides arebetween about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.The optimal length can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl)glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agarwal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisense phytasesequences of the invention (see, e.g., Gold (1995) J. of Biol. Chem.270:13581-13584).

Inhibitory Ribozymes

The invention provides ribozymes comprising the new phytase sequencemodifications of the invention, where the ribozymes of the invention arecapable of binding phytase message which can inhibit phytase enzymeactivity by targeting mRNA. Strategies for designing ribozymes andselecting the phytase-specific antisense sequence for targeting are welldescribed in the scientific and patent literature, and the skilledartisan can design such ribozymes using the novel reagents of theinvention. Ribozymes act by binding to a target RNA through the targetRNA binding portion of a ribozyme which is held in close proximity to anenzymatic portion of the RNA that cleaves the target RNA. Thus, theribozyme recognizes and binds a target RNA through complementarybase-pairing, and once bound to the correct site, acts enzymatically tocleave and inactivate the target RNA. Cleavage of a target RNA in such amanner will destroy its ability to direct synthesis of an encodedprotein if the cleavage occurs in the coding sequence. After a ribozymehas bound and cleaved its RNA target, it is typically released from thatRNA and so can bind and cleave new targets repeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

The enzymatic ribozyme RNA molecule can be formed in a hammerhead motif,but may also be formed in the motif of a hairpin, hepatitis delta virus,group I intron or RNaseP-like RNA (in association with an RNA guidesequence). Examples of such hammerhead motifs are described by Rossi(1992) Aids Research and Human Retroviruses 8:183; hairpin motifs byHampel (1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res.18:299; the hepatitis delta virus motif by Perrotta (1992) Biochemistry31:16; the RNaseP motif by Guerrier-Takada (1983) Cell 35:849; and thegroup I intron by Cech U.S. Pat. No. 4,987,071. The recitation of thesespecific motifs is not intended to be limiting; those skilled in the artwill recognize that an enzymatic RNA molecule of this invention has aspecific substrate binding site complementary to one or more of thetarget gene RNA regions, and has nucleotide sequence within orsurrounding that substrate binding site which imparts an RNA cleavingactivity to the molecule.

RNA Interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising an enzyme sequence of theinvention. The RNAi molecule comprises a double-stranded RNA (dsRNA)molecule. The RNAi molecule, e.g., siRNA and/or miRNA, can inhibitexpression of phytase enzyme gene. In one aspect, the RNAi molecule,e.g., siRNA and/or miRNA, is about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25 or more duplex nucleotides in length.

While the invention is not limited by any particular mechanism ofaction, the RNAi can enter a cell and cause the degradation of asingle-stranded RNA (ssRNA) of similar or identical sequences, includingendogenous mRNAs. When a cell is exposed to double-stranded RNA (dsRNA),mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi). A possible basic mechanism behind RNAiis the breaking of a double-stranded RNA (dsRNA) matching a specificgene sequence into short pieces called short interfering RNA, whichtrigger the degradation of mRNA that matches its sequence. In oneaspect, the RNAi's of the invention are used in gene-silencingtherapeutics, see, e.g., Shuey (2002) Drug Discov. Today 7:1040-1046. Inone aspect, the invention provides methods to selectively degrade RNAusing the RNAi's molecules, e.g., siRNA and/or miRNA, of the invention.In one aspect, the micro-inhibitory RNA (miRNA) inhibits translation,and the siRNA inhibits transcription. The process may be practiced invitro, ex vivo or in vivo. In one aspect, the RNAi molecules of theinvention can be used to generate a loss-of-function mutation in a cell,an organ or an animal. Methods for making and using RNAi molecules,e.g., siRNA and/or miRNA, for selectively degrade RNA are well known inthe art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824; 6,515,109;6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleicacids of the invention, e.g., those encoding a phytase enzyme. Thesemethods can be repeated or used in various combinations to generatephytase enzymes having an altered or different activity or an altered ordifferent stability from that of a phytase encoded by the templatenucleic acid. These methods also can be repeated or used in variouscombinations, e.g., to generate variations in gene/message expression,message translation or message stability. In another aspect, the geneticcomposition of a cell is altered by, e.g., modification of a homologousgene ex vivo, followed by its reinsertion into the cell.

The invention also provides methods for changing the characteristics ofa phytase of the invention by mutagenesis and other method, includingdirected evolution, e.g., Diversa Corporation's (San Diego, Calif.)proprietary approaches; e.g., DirectEvolution; (see, e.g., U.S. Pat. No.5,830,696; Gene Site Saturation Mutagenesis (GSSM) (see, e.g., U.S. Pat.Nos. 6,171,820 and 6,579,258), Exonuclease-Mediated Gene Assembly inDirected Evolution (see, e.g., U.S. Pat. Nos. 6,361,974 and 6,352,842),End Selection in Directed Evolution (see, e.g., U.S. Pat. Nos. 6,358,709and 6,238,884), Recombination-Based Synthesis Shuffling (see, e.g., U.S.Pat. Nos. 5,965,408 and 6,440,668, and Australian Patent No. AU724521),and Directed Evolution of Thermophilic Enzymes (see, e.g., U.S. Pat.Nos. 5,830,696 and 6,335,179).

In one aspect, the characteristics of a phytase are modified by aDirectEvolution protocol comprising: a) the subjection of one or moremolecular templates, e.g., the phytase nucleic acids of the invention,to mutagenesis to generate novel molecules, and b) the selection amongthese progeny species of novel molecules with more desirablecharacteristics. The power of directed evolution depends on the startingchoice of starting templates (e.g., the “parent” SEQ ID NO:1, or anysequence of this invention), as well as on the mutagenesis process(es)chosen and the screening process(es) used. Thus, the invention providesnovel highly active, physiologically effective, and economical sourcesof phytase activity, including novel phytases that: a) have superioractivities under one or more specific applications, such as hightemperature manufacture of foodstuffs, and are thus useful foroptimizing these specific applications; b) are useful as templates fordirected evolution to achieve even further improved novel molecules; andc) are useful as tools for the identification of additional relatedmolecules by means such as hybridization-based approaches.

A nucleic acid of the invention can be altered by any means. Forexample, random or stochastic methods, or, non-stochastic, or “directedevolution,” methods. Methods for random mutation of genes are well knownin the art, see, e.g., U.S. Pat. No. 5,830,696. For example, mutagenscan be used to randomly mutate a gene. Mutagens include, e.g.,ultraviolet light or gamma irradiation, or a chemical mutagen, e.g.,mitomycin, nitrous acid, photoactivated psoralens, alone or incombination, to induce DNA breaks amenable to repair by recombination.Other chemical mutagens include, for example, sodium bisulfite, nitrousacid, hydroxylamine, hydrazine or formic acid. Other mutagens areanalogues of nucleotide precursors, e.g., nitrosoguanidine,5-bromouracil, 2-aminopurine, or acridine. These agents can be added toa PCR reaction in place of the nucleotide precursor thereby mutating thesequence. Intercalating agents such as proflavine, acriflavine,quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCRmutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleicacids, e.g., genes, can be reassembled after random, or “stochastic,”fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862;6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. Inalternative aspects, modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, genesite saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR),recombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation, and/or acombination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods ofthe invention: Stemmer (1999) “Molecular breeding of viruses fortargeting and other clinical properties” Tumor Targeting 4:1-4; Ness(1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians (1999) “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang (1997) “Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening” Proc. Natl. Acad.Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Crameri et al. (1996) “Improved green fluorescent protein bymolecular evolution using DNA shuffling” Nature Biotechnology14:315-319; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmidform large numbers of oligodeoxyribonucleotides” Gene, 164:49-53;Stemmer (1995) “The Evolution of Molecular Computation” Science 270:1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNAshuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller & Smith (1987)“Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765-8787 (1985); Nakamaye (1986) “Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 14: 9679-9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791-802; and Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols used in the methods of the invention include pointmismatch repair (Kramer (1984) “Point Mismatch Repair” Cell 38:879-887),mutagenesis using repair-deficient host strains (Carter et al. (1985)“Improved oligonucleotide site-directed mutagenesis using M13 vectors”Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites” Gene 34:315-323; and Grundstrom etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’ gene synthesis” Nucl. Acids Res. 13: 3305-3316),double-strand break repair (Mandecki (1986); Arnold (1993) “Proteinengineering for unusual environments” Current Opinion in Biotechnology4:450-455. “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA 83:7177-7181).

Additional details or alternative protocols on many of the above methodscan be found in Methods in Enzymology Volume 154, which also describesuseful controls for trouble-shooting problems with various mutagenesismethods. See also U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997),“Methods for In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmeret al. (Sep. 22, 1998) “Methods for Generating Polynucleotides havingDesired Characteristics by Iterative Selection and Recombination;” U.S.Pat. No. 5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis byRandom Fragmentation and Reassembly;” U.S. Pat. No. 5,834,252 toStemmer, et al. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;”U.S. Pat. No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methodsand Compositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImLmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

U.S. applications provide additional details or alternative protocolsregarding various diversity generating methods, including “SHUFFLING OFCODON ALTERED GENES” by Patten et al. filed Sep. 28, 1999, (U.S. Ser.No. 09/407,800); “EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVESEQUENCE RECOMBINATION” by del Cardayre et al., filed Jul. 15, 1998(U.S. Ser. No. 09/166,188), and Jul. 15, 1999 (U.S. Ser. No.09/354,922); “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” byCrameri et al., filed Sep. 28, 1999 (U.S. Ser. No. 09/408,392), and“OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al.,filed Jan. 18, 2000 (PCT/US00/01203); “USE OF CODON-VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al.,filed Sep. 28, 1999 (U.S. Ser. No. 09/408,393); “METHODS FOR MAKINGCHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIREDCHARACTERISTICS” by Selifonov et al., filed Jan. 18, 2000,(PCT/US00/01202) and, e.g. “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jul. 18, 2000 (U.S. Ser. No. 09/618,579);“METHODS OF POPULATING DATA STRUCTURES FOR USE IN EVOLUTIONARYSIMULATIONS” by Selifonov and Stemmer, filed Jan. 18, 2000(PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACID TEMPLATE-MEDIATEDRECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” by Affholter, filedSep. 6, 2000 (U.S. Ser. No. 09/656,549).

Non-stochastic, or “directed evolution,” methods include, e.g., genesite saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR),or a combination thereof are used to modify the nucleic acids of theinvention to generate phytases with new or altered properties (e.g.,activity under highly acidic or alkaline conditions, high or lowtemperatures, and the like). Polypeptides encoded by the modifiednucleic acids can be screened for an activity before testing for aphytase or other activity. Any testing modality or protocol can be used,e.g., using a capillary array platform. See, e.g., U.S. Pat. Nos.6,361,974; 6,280,926; 5,939,250.

Saturation Mutagenesis, or GSSM

The invention also provides methods for making enzyme using Gene SiteSaturation mutagenesis, or, GSSM, as described herein, and also in U.S.Pat. Nos. 6,171,820 and 6,579,258.

In one aspect, codon primers containing a degenerate N,N,G/T sequenceare used to introduce point mutations into a polynucleotide, e.g., aphytase enzyme or an antibody of the invention, so as to generate a setof progeny polypeptides in which a full range of single amino acidsubstitutions is represented at each amino acid position, e.g., an aminoacid residue in an enzyme active site or ligand binding site targeted tobe modified. These oligonucleotides can comprise a contiguous firsthomologous sequence, a degenerate N,N,G/T sequence, and, optionally, asecond homologous sequence. The downstream progeny translationalproducts from the use of such oligonucleotides include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,G/T sequence includes codons for all20 amino acids. In one aspect, one such degenerate oligonucleotide(comprising, e.g., one degenerate N,N,G/T cassette) is used forsubjecting each original codon in a parental polynucleotide template toa full range of codon substitutions. In another aspect, at least twodegenerate cassettes are used—either in the same oligonucleotide or not,for subjecting at least two original codons in a parental polynucleotidetemplate to a full range of codon substitutions. For example, more thanone N,N,G/T sequence can be contained in one oligonucleotide tointroduce amino acid mutations at more than one site. This plurality ofN,N,G/T sequences can be directly contiguous, or separated by one ormore additional nucleotide sequence(s). In another aspect,oligonucleotides serviceable for introducing additions and deletions canbe used either alone or in combination with the codons containing anN,N,G/T sequence, to introduce any combination or permutation of aminoacid additions, deletions, and/or substitutions.

In one aspect, simultaneous mutagenesis of two or more contiguous aminoacid positions is done using an oligonucleotide that contains contiguousN,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In anotheraspect, degenerate cassettes having less degeneracy than the N,N,G/Tsequence are used. For example, it may be desirable in some instances touse (e.g. in an oligonucleotide) a degenerate triplet sequencecomprising only one N, where said N can be in the first second or thirdposition of the triplet. Any other bases including any combinations andpermutations thereof can be used in the remaining two positions of thetriplet. Alternatively, it may be desirable in some instances to use(e.g. in an oligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)allows for systematic and easy generation of a full range of possiblenatural amino acids (for a total of 20 amino acids) into each and everyamino acid position in a polypeptide (in alternative aspects, themethods also include generation of less than all possible substitutionsper amino acid residue, or codon, position). For example, for a 100amino acid polypeptide, 2000 distinct species (i.e. 20 possible aminoacids per position X 100 amino acid positions) can be generated. Throughthe use of an oligonucleotide or set of oligonucleotides containing adegenerate N,N,G/T triplet, 32 individual sequences can code for all 20possible natural amino acids. Thus, in a reaction vessel in which aparental polynucleotide sequence is subjected to saturation mutagenesisusing at least one such oligonucleotide, there are generated 32 distinctprogeny polynucleotides encoding 20 distinct polypeptides. In contrast,the use of a non-degenerate oligonucleotide in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.Nondegenerate oligonucleotides can optionally be used in combinationwith degenerate primers disclosed; for example, nondegenerateoligonucleotides can be used to generate specific point mutations in aworking polynucleotide. This provides one means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel containspolynucleotides encoding at least 20 progeny polypeptide (e.g., phytaseenzymes) molecules such that all 20 natural amino acids are representedat the one specific amino acid position corresponding to the codonposition mutagenized in the parental polynucleotide (other aspects useless than all 20 natural combinations). The 32-fold degenerate progenypolypeptides generated from each saturation mutagenesis reaction vesselcan be subjected to clonal amplification (e.g. cloned into a suitablehost, e.g., E. coli host, using, e.g., an expression vector) andsubjected to expression screening. When an individual progenypolypeptide is identified by screening to display a favorable change inproperty (when compared to the parental polypeptide, such as increasedglucan hydrolysis activity under alkaline or acidic conditions), it canbe sequenced to identify the correspondingly favorable amino acidsubstitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in aparental polypeptide using saturation mutagenesis as disclosed herein,favorable amino acid changes may be identified at more than one aminoacid position. One or more new progeny molecules can be generated thatcontain a combination of all or part of these favorable amino acidsubstitutions. For example, if 2 specific favorable amino acid changesare identified in each of 3 amino acid positions in a polypeptide, thepermutations include 3 possibilities at each position (no change fromthe original amino acid, and each of two favorable changes) and 3positions. Thus, there are 3×3×3 or 27 total possibilities, including 7that were previously examined—6 single point mutations (i.e. 2 at eachof three positions) and no change at any position.

In yet another aspect, site-saturation mutagenesis can be used togetherwith shuffling, chimerization, recombination and other mutagenizingprocesses, along with screening. This invention provides for the use ofany mutagenizing process(es), including saturation mutagenesis, in aniterative manner. In one exemplification, the iterative use of anymutagenizing process(es) is used in combination with screening.

The invention also provides for the use of proprietary codon primers(containing a degenerate N,N,N sequence) to introduce point mutationsinto a polynucleotide, so as to generate a set of progeny polypeptidesin which a full range of single amino acid substitutions is representedat each amino acid position (Gene Site Saturation Mutagenesis (GSSM)).The oligos used are comprised contiguously of a first homologoussequence, a degenerate N,N,N sequence and in one aspect but notnecessarily a second homologous sequence. The downstream progenytranslational products from the use of such oligos include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,N sequence includes codons for all 20amino acids.

In one aspect, one such degenerate oligo (comprising one degenerateN,N,N cassette) is used for subjecting each original codon in a parentalpolynucleotide template to a full range of codon substitutions. Inanother aspect, at least two degenerate N,N,N cassettes are used—eitherin the same oligo or not, for subjecting at least two original codons ina parental polynucleotide template to a full range of codonsubstitutions. Thus, more than one N,N,N sequence can be contained inone oligo to introduce amino acid mutations at more than one site. Thisplurality of N,N,N sequences can be directly contiguous, or separated byone or more additional nucleotide sequence(s). In another aspect, oligosserviceable for introducing additions and deletions can be used eitheralone or in combination with the codons containing an N,N,N sequence, tointroduce any combination or permutation of amino acid additions,deletions and/or substitutions.

In a particular exemplification, it is possible to simultaneouslymutagenize two or more contiguous amino acid positions using an oligothat contains contiguous N,N,N triplets, i.e. a degenerate (N,N,N)_(n)sequence.

In another aspect, the present invention provides for the use ofdegenerate cassettes having less degeneracy than the N,N,N sequence. Forexample, it may be desirable in some instances to use (e.g. in an oligo)a degenerate triplet sequence comprising only one N, where the N can bein the first second or third position of the triplet. Any other basesincluding any combinations and permutations thereof can be used in theremaining two positions of the triplet. Alternatively, it may bedesirable in some instances to use (e.g., in an oligo) a degenerateN,N,N triplet sequence, N,N,G/T, or an N,N, G/C triplet sequence.

It is appreciated, however, that the use of a degenerate triplet (suchas N,N,G/T or an N,N, G/C triplet sequence) as disclosed in the instantinvention is advantageous for several reasons. In one aspect, thisinvention provides a means to systematically and fairly easily generatethe substitution of the full range of possible amino acids (for a totalof 20 amino acids) into each and every amino acid position in apolypeptide. Thus, for a 100 amino acid polypeptide, the inventionprovides a way to systematically and fairly easily generate 2000distinct species (i.e., 20 possible amino acids per position times 100amino acid positions). It is appreciated that there is provided, throughthe use of an oligo containing a degenerate N,N,G/T or an N,N, G/Ctriplet sequence, 32 individual sequences that code for 20 possibleamino acids. Thus, in a reaction vessel in which a parentalpolynucleotide sequence is subjected to saturation mutagenesis using onesuch oligo, there are generated 32 distinct progeny polynucleotidesencoding 20 distinct polypeptides. In contrast, the use of anon-degenerate oligo in site-directed mutagenesis leads to only oneprogeny polypeptide product per reaction vessel.

This invention also provides for the use of nondegenerate oligos, whichcan optionally be used in combination with degenerate primers disclosed.It is appreciated that in some situations, it is advantageous to usenondegenerate oligos to generate specific point mutations in a workingpolynucleotide. This provides a means to generate specific silent pointmutations, point mutations leading to corresponding amino acid changesand point mutations that cause the generation of stop codons and thecorresponding expression of polypeptide fragments.

Thus, in one aspect of this invention, each saturation mutagenesisreaction vessel contains polynucleotides encoding at least 20 progenypolypeptide molecules such that all 20 amino acids are represented atthe one specific amino acid position corresponding to the codon positionmutagenized in the parental polynucleotide. The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g., cloned into asuitable E. coli host using an expression vector) and subjected toexpression screening. When an individual progeny polypeptide isidentified by screening to display a favorable change in property (whencompared to the parental polypeptide), it can be sequenced to identifythe correspondingly favorable amino acid substitution contained therein.

It is appreciated that upon mutagenizing each and every amino acidposition in a parental polypeptide using saturation mutagenesis asdisclosed herein, favorable amino acid changes may be identified at morethan one amino acid position. One or more new progeny molecules can begenerated that contain a combination of all or part of these favorableamino acid substitutions. For example, if 2 specific favorable aminoacid changes are identified in each of 3 amino acid positions in apolypeptide, the permutations include 3 possibilities at each position(no change from the original amino acid and each of two favorablechanges) and 3 positions. Thus, there are 3×3×3 or 27 totalpossibilities, including 7 that were previously examined—6 single pointmutations (i.e., 2 at each of three positions) and no change at anyposition.

Thus, in a non-limiting exemplification, this invention provides for theuse of saturation mutagenesis in combination with additionalmutagenization processes, such as process where two or more relatedpolynucleotides are introduced into a suitable host cell such that ahybrid polynucleotide is generated by recombination and reductivereassortment.

In addition to performing mutagenesis along the entire sequence of agene, the instant invention provides that mutagenesis can be use toreplace each of any number of bases in a polynucleotide sequence,wherein the number of bases to be mutagenized is in one aspect everyinteger from 15 to 100,000. Thus, instead of mutagenizing every positionalong a molecule, one can subject every or a discrete number of bases(in one aspect a subset totaling from 15 to 100,000) to mutagenesis. Inone aspect, a separate nucleotide is used for mutagenizing each positionor group of positions along a polynucleotide sequence. A group of 3positions to be mutagenized may be a codon. The mutations can beintroduced using a mutagenic primer, containing a heterologous cassette,also referred to as a mutagenic cassette. Exemplary cassettes can havefrom 1 to 500 bases. Each nucleotide position in such heterologouscassettes be N, A, C, G, T, A/C, A/G, A/T, C/G, CT, G/T, C/G/T, A/G/T,A/C/T, A/C/G, or E, where E is any base that is not A, C, G, or T (E canbe referred to as a designer oligo).

In a general sense, saturation mutagenesis is comprised of mutagenizinga complete set of mutagenic cassettes (wherein each cassette is in oneaspect about 1-500 bases in length) in defined polynucleotide sequenceto be mutagenized (wherein the sequence to be mutagenized is in oneaspect from about 15 to 100,000 bases in length). Thus, a group ofmutations (ranging from 1 to 100 mutations) is introduced into eachcassette to be mutagenized. A grouping of mutations to be introducedinto one cassette can be different or the same from a second grouping ofmutations to be introduced into a second cassette during the applicationof one round of saturation mutagenesis. Such groupings are exemplifiedby deletions, additions, groupings of particular codons and groupings ofparticular nucleotide cassettes.

Defined sequences to be mutagenized include a whole gene, pathway, cDNA,an entire open reading frame (ORF) and entire promoter, enhancer,repressor/transactivator, origin of replication, intron, operator, orany polynucleotide functional group. Generally, a “defined sequences”for this purpose may be any polynucleotide that a 15 base-polynucleotidesequence and polynucleotide sequences of lengths between 15 bases and15,000 bases (this invention specifically names every integer inbetween). Considerations in choosing groupings of codons include typesof amino acids encoded by a degenerate mutagenic cassette.

In one exemplification a grouping of mutations that can be introducedinto a mutagenic cassette, this invention specifically provides fordegenerate codon substitutions (using degenerate oligos) that code for2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20amino acids at each position and a library of polypeptides encodedthereby.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate polypeptides, e.g., phytase enzymes or antibodiesof the invention, with new or altered properties. SLR is a method ofligating oligonucleotide fragments together non-stochastically. Thismethod differs from stochastic oligonucleotide shuffling in that thenucleic acid building blocks are not shuffled, concatenated orchimerized randomly, but rather are assembled non-stochastically. See,e.g., U.S. Pat. Nos. 6,773,900; 6,740,506; 6,713,282; 6,635,449;6,605,449; 6,537,776.

In one aspect, SLR comprises the following steps: (a) providing atemplate polynucleotide, wherein the template polynucleotide comprisessequence encoding a homologous gene; (b) providing a plurality ofbuilding block polynucleotides, wherein the building blockpolynucleotides are designed to cross-over reassemble with the templatepolynucleotide at a predetermined sequence, and a building blockpolynucleotide comprises a sequence that is a variant of the homologousgene and a sequence homologous to the template polynucleotide flankingthe variant sequence; (c) combining a building block polynucleotide witha template polynucleotide such that the building block polynucleotidecross-over reassembles with the template polynucleotide to generatepolynucleotides comprising homologous gene sequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprising over 10¹⁰⁰ different chimeras. SLR can be used to generatelibraries comprising over 10¹⁰⁰⁰ different progeny chimeras. Thus,aspects of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In one aspect, the annealed building pieces aretreated with an enzyme, such as a ligase (e.g. T4 DNA ligase), toachieve covalent bonding of the building pieces. In one aspect, anon-stochastic method termed synthetic ligation reassembly (SLR), thatis somewhat related to stochastic shuffling, save that the nucleic acidbuilding blocks are not shuffled or concatenated or chimerized randomly,but rather are assembled non-stochastically can be used to createvariants.

The SLR method does not depend on the presence of a high level ofhomology between polynucleotides to be shuffled. The invention can beused to non-stochastically generate libraries (or sets) of progenymolecules comprising over 10¹⁰⁰ different chimeras. Conceivably, SLR caneven be used to generate libraries comprising over 10¹⁰⁰⁰ differentprogeny chimeras.

Thus, in one aspect, the invention provides a non-stochastic method ofproducing a set of finalized chimeric nucleic acid molecules having anoverall assembly order that is chosen by design, which method iscomprises the steps of generating by design a plurality of specificnucleic acid building blocks having serviceable mutually compatibleligatable ends, and assembling these nucleic acid building blocks, suchthat a designed overall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, in one aspect, the overall assembly order inwhich the nucleic acid building blocks can be coupled is specified bythe design of the ligatable ends and, if more than one assembly step isto be used, then the overall assembly order in which the nucleic acidbuilding blocks can be coupled is also specified by the sequential orderof the assembly step(s). In one aspect of the invention, the annealedbuilding pieces are treated with an enzyme, such as a ligase (e.g., T4DNA ligase) to achieve covalent bonding of the building pieces.

In a another aspect, the design of nucleic acid building blocks isobtained upon analysis of the sequences of a set of progenitor nucleicacid templates that serve as a basis for producing a progeny set offinalized chimeric nucleic acid molecules. These progenitor nucleic acidtemplates thus serve as a source of sequence information that aids inthe design of the nucleic acid building blocks that are to bemutagenized, i.e. chimerized or shuffled.

In one exemplification, the invention provides for the chimerization ofa family of related genes and their encoded family of related products.In a particular exemplification, the encoded products are enzymes.Enzymes and polypeptides for use in the invention can be mutagenized inaccordance with the methods described herein.

Thus according to one aspect of the invention, the sequences of aplurality of progenitor nucleic acid templates are aligned in order toselect one or more demarcation points, which demarcation points can belocated at an area of homology. The demarcation points can be used todelineate the boundaries of nucleic acid building blocks to begenerated. Thus, the demarcation points identified and selected in theprogenitor molecules serve as potential chimerization points in theassembly of the progeny molecules.

Typically a serviceable demarcation point is an area of homology(comprised of at least one homologous nucleotide base) shared by atleast two progenitor templates, but the demarcation point can be an areaof homology that is shared by at least half of the progenitor templates,at least two thirds of the progenitor templates, at least three fourthsof the progenitor templates, or almost all of the progenitor templates.In one aspect, a serviceable demarcation point is an area of homologythat is shared by all of the progenitor templates.

In one aspect, the ligation reassembly process is performed exhaustivelyin order to generate an exhaustive library. In other words, all possibleordered combinations of the nucleic acid building blocks are representedin the set of finalized chimeric nucleic acid molecules. At the sametime, the assembly order (i.e. the order of assembly of each buildingblock in the 5′ to 3 sequence of each finalized chimeric nucleic acid)in each combination is by design (or non-stochastic). Because of thenon-stochastic nature of the method, the possibility of unwanted sideproducts is greatly reduced.

In another aspect, the method provides that, the ligation reassemblyprocess is performed systematically, for example in order to generate asystematically compartmentalized library, with compartments that can bescreened systematically, e.g., one by one. In other words the inventionprovides that, through the selective and judicious use of specificnucleic acid building blocks, coupled with the selective and judicioususe of sequentially stepped assembly reactions, an experimental designcan be achieved where specific sets of progeny products are made in eachof several reaction vessels. This allows a systematic examination andscreening procedure to be performed. Thus, it allows a potentially verylarge number of progeny molecules to be examined systematically insmaller groups.

Because of its ability to perform chimerizations in a manner that ishighly flexible yet exhaustive and systematic as well, particularly whenthere is a low level of homology among the progenitor molecules, theinstant invention provides for the generation of a library (or set)comprised of a large number of progeny molecules. Because of thenon-stochastic nature of the instant ligation reassembly invention, theprogeny molecules generated can comprise a library of finalized chimericnucleic acid molecules having an overall assembly order that is chosenby design. In a particularly aspect, such a generated library iscomprised of greater than 10³ to greater than 10¹⁰⁰⁰ different progenymolecular species.

In one aspect, a set of finalized chimeric nucleic acid molecules,produced as described is comprised of a polynucleotide encoding apolypeptide. According to one aspect, this polynucleotide is a gene,which may be a man-made gene. According to another aspect, thispolynucleotide is a gene pathway, which may be a man-made gene pathway.The invention provides that one or more man-made genes generated by theinvention may be incorporated into a man-made gene pathway, such aspathway operable in a eukaryotic organism (including a plant).

In another exemplification, the synthetic nature of the step in whichthe building blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (e.g., by mutagenesis) or in an in vivoprocess (e.g., by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

Thus, according to another aspect, the invention provides that a nucleicacid building block can be used to introduce an intron. Thus, theinvention provides that functional introns may be introduced into aman-made gene of the invention. The invention also provides thatfunctional introns may be introduced into a man-made gene pathway of theinvention. Accordingly, the invention provides for the generation of achimeric polynucleotide that is a man-made gene containing one (or more)artificially introduced intron(s).

Accordingly, the invention also provides for the generation of achimeric polynucleotide that is a man-made gene pathway containing one(or more) artificially introduced intron(s). In one aspect, theartificially introduced intron(s) are functional in one or more hostcells for gene splicing much in the way that naturally-occurring intronsserve functionally in gene splicing. The invention provides a process ofproducing man-made intron-containing polynucleotides to be introducedinto host organisms for recombination and/or splicing.

A man-made gene produced using the invention can also serve as asubstrate for recombination with another nucleic acid. Likewise, aman-made gene pathway produced using the invention can also serve as asubstrate for recombination with another nucleic acid. In one aspect,the recombination is facilitated by, or occurs at, areas of homologybetween the man-made intron-containing gene and a nucleic acid withserves as a recombination partner. In one aspect, the recombinationpartner may also be a nucleic acid generated by the invention, includinga man-made gene or a man-made gene pathway. Recombination may befacilitated by or may occur at areas of homology that exist at the one(or more) artificially introduced intron(s) in the man-made gene.

The synthetic ligation reassembly method of the invention utilizes aplurality of nucleic acid building blocks, each of which can have twoligatable ends. The two ligatable ends on each nucleic acid buildingblock may be two blunt ends (i.e. each having an overhang of zeronucleotides), or one blunt end and one overhang, or two overhangs.

A useful overhang for this purpose may be a 3′ overhang or a 5′overhang. Thus, a nucleic acid building block may have a 3′ overhang oralternatively a 5′ overhang or alternatively two 3′ overhangs oralternatively two 5′ overhangs. The overall order in which the nucleicacid building blocks are assembled to form a finalized chimeric nucleicacid molecule is determined by purposeful experimental design and is notrandom.

In one aspect, a nucleic acid building block is generated by chemicalsynthesis of two single-stranded nucleic acids (also referred to assingle-stranded oligos) and contacting them so as to allow them toanneal to form a double-stranded nucleic acid building block.

A double-stranded nucleic acid building block can be of variable size.The sizes of these building blocks can be small or large. Exemplarysizes for building block range from 1 base pair (not including anyoverhangs) to 100,000 base pairs (not including any overhangs). Othersize ranges are also provided, which have lower limits of from 1 bp to10,000 bp (including every integer value in between), and upper limitsof from 2 bp to 100,000 bp (including every integer value in between).

Many methods exist by which a double-stranded nucleic acid buildingblock can be generated that is serviceable for the invention; and theseare known in the art and can be readily performed by the skilledartisan.

According to one aspect, a double-stranded nucleic acid building blockis generated by first generating two single stranded nucleic acids andallowing them to anneal to form a double-stranded nucleic acid buildingblock. The two strands of a double-stranded nucleic acid building blockmay be complementary at every nucleotide apart from any that form anoverhang; thus containing no mismatches, apart from any overhang(s).According to another aspect, the two strands of a double-strandednucleic acid building block are complementary at fewer than everynucleotide apart from any that form an overhang. Thus, according to thisaspect, a double-stranded nucleic acid building block can be used tointroduce codon degeneracy. In one aspect, the codon degeneracy isintroduced using the site-saturation mutagenesis described herein, usingone or more N,N,G/T cassettes or alternatively using one or more N,N,Ncassettes.

The in vivo recombination method of the invention can be performedblindly on a pool of unknown hybrids or alleles of a specificpolynucleotide or sequence. However, it is not necessary to know theactual DNA or RNA sequence of the specific polynucleotide.

The approach of using recombination within a mixed population of genescan be useful for the generation of any useful proteins, for example,interleukin I, antibodies, tPA and growth hormone. This approach may beused to generate proteins having altered specificity or activity. Theapproach may also be useful for the generation of hybrid nucleic acidsequences, for example, promoter regions, introns, exons, enhancersequences, 31 untranslated regions or 51 untranslated regions of genes.Thus this approach may be used to generate genes having increased ratesof expression. This approach may also be useful in the study ofrepetitive DNA sequences. Finally, this approach may be useful to mutateribozymes or aptamers.

In one aspect variants of the polynucleotides and polypeptides describedherein are obtained by the use of repeated cycles of reductivereassortment, recombination and selection which allow for the directedmolecular evolution of highly complex linear sequences, such as DNA, RNAor proteins thorough recombination.

In vivo shuffling of molecules is useful in providing variants and canbe performed utilizing the natural property of cells to recombinemultimers. While recombination in vivo has provided the major naturalroute to molecular diversity, genetic recombination remains a relativelycomplex process that involves 1) the recognition of homologies; 2)strand cleavage, strand invasion, and metabolic steps leading to theproduction of recombinant chiasma; and finally 3) the resolution ofchiasma into discrete recombined molecules. The formation of the chiasmarequires the recognition of homologous sequences.

In another aspect, the invention includes a method for producing ahybrid polynucleotide from at least a first polynucleotide and a secondpolynucleotide. The invention can be used to produce a hybridpolynucleotide by introducing at least a first polynucleotide and asecond polynucleotide which share at least one region of partialsequence homology (e.g., SEQ ID NO:1) into a suitable host cell. Theregions of partial sequence homology promote processes that result insequence reorganization producing a hybrid polynucleotide. The term“hybrid polynucleotide”, as used herein, is any nucleotide sequencewhich results from the method of the present invention and containssequence from at least two original polynucleotide sequences. Suchhybrid polynucleotides can result from intermolecular recombinationevents which promote sequence integration between DNA molecules. Inaddition, such hybrid polynucleotides can result from intramolecularreductive reassortment processes which utilize repeated sequences toalter a nucleotide sequence within a DNA molecule.

The invention provides methods for generating hybrid polynucleotideswhich may encode biologically active hybrid polypeptides (e.g., a hybridphytase). In one aspect, the original polynucleotides encodebiologically active polypeptides. The method of the invention producesnew hybrid polypeptides by utilizing cellular processes which integratethe sequence of the original polynucleotides such that the resultinghybrid polynucleotide encodes a polypeptide demonstrating activitiesderived from the original biologically active polypeptides. For example,the original polynucleotides may encode a particular enzyme fromdifferent microorganisms. An enzyme encoded by a first polynucleotidefrom one organism or variant may, for example, function effectivelyunder a particular environmental condition, e.g., high salinity. Anenzyme encoded by a second polynucleotide from a different organism orvariant may function effectively under a different environmentalcondition, such as extremely high temperatures. A hybrid polynucleotidecontaining sequences from the first and second original polynucleotidesmay encode an enzyme which exhibits characteristics of both enzymesencoded by the original polynucleotides. Thus, the enzyme encoded by thehybrid polynucleotide may function effectively under environmentalconditions shared by each of the enzymes encoded by the first and secondpolynucleotides, e.g., high salinity and extreme temperatures.

In addition to the various methods described above, various methods areknown in the art that can be used to obtain hybrid polynucleotides withenhanced enzymatic properties. The following examples illustrate the useof such procedures for obtaining thermostable or thermotolerant enzymesby mutagenesis of a polynucleotide encoding a wild-type enzyme ofinterest.

For example, in one aspect, the invention uses methods as described byM. Lehmann et al. (in Biochimica et Biophysica Acta 1543:408-415, 2000)describes a “consensus approach” wherein sequence alignment ofhomologous fungal phytases was used to calculate a consensus phytaseamino acid sequence. Upon construction of the corresponding consensusgen, recombinant expression and purification, the recombinant phytaseobtained displayed an unfolding temperature (Tm) 15-22° C. higher thanthat of all parent phytases used in the design. Site-directedmutagenesis of the gene encoding the recombinant protein was used tofurther increase the Tm value to 90.4° C. The thermostabilizing effectwas attributed to a combination of multiple amino acid exchanges thatwere distributed over the entire sequence of the protein and mainlyaffected surface-exposed residues.

In one aspect, the invention uses methods to obtaining an enzyme withenhanced thermal properties as described by L. Jermutus et al. (J. ofBiotechnology 85:15-24, 2001). In this approach ionic interactions andhydrogen bonds on the surface of Aspergillus terreus phytase were firstrestored to correspond to those present in the homologous, but morethermostable enzyme from A. niger. Then entire secondary structuralelements were replaced in the same region and based on the crystalstructure of A. niger phytase. The replacement of one l-helix on thesurface of A. terreus phytase by the corresponding stretch of A nigerphytase resulted in a structure-based chimeric enzyme (fusion protein)with improved thermostability and unaltered enzymatic activity.

In one aspect, the invention uses methods as described by L. Giver etal. (Proc. Natl. Acad. Sci. USA 95:12809-12813, 1998), who describes aprocedure wherein six generations of random mutagenesis introducedduring mutagenic PCR of a polynucleotide encoding Bacillus subtilisp-nitrobenzyl esterase followed by in vitro recombination based on themethod of Stemmer resulted in a recombinant esterase with increasedthermostability (greater than 14° C. increase in Tm) withoutcompromising catalytic activity at lower temperatures.

In one aspect, the invention uses methods as described by C. Vetriani etal. (Proc. Natl. Acad. Sci USA 95:12300-12305, 1998), who describes aprocedure by which homology-based modeling and direct structurecomparison of the hexameric glutamate dehydrogenases from thehyperthermophiles Pyrococcus furiosus and Thermococcus litoralis, withoptimal growth temperatures of 100° C. and 88° C., respectively, wereused to determine key thermostabilizing features. An intersubunition-pair network observed to be substantially reduced in the less stableenzyme was altered by mutagenesis of two residues therein to restore theinteractions found in the more stable enzyme. Although either singlemutation had adverse effects on the thermostability, with both mutationsin place, a four-fold improvement of stability at 104° C. over thewild-type enzyme was observed.

In one aspect, the invention uses methods as described by A. Tomschy etal. (Protein Science 9:1304-1311, 2000), who describes a procedureutilizing the crystal structure of Aspergillus Niger phytase (at 2.5angstroms resolution) to specify all active sites of the enzyme. Amultiple amino acid sequence alignment was then used to identifynon-conserved active site residues that might correlate with a givenfavorable property of interest. Using this approach, Gln27 of A.fumigatus phytase, which differed from Leu27 of A. niger, was identifiedas likely to be involved in substrate binding and/or release andresponsible for the lower specific activity of the A. fumigatus phytase(26.5 vs. 196 6 U/mg protein at pH 5.0). Site directed mutagenesis ofGln27 of A. fumigatus phytase to Leu increased the specific activity ofthe mutant enzyme to 92.1 U/mg protein.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide, an expression cassette, cloning mechanism or vectorof the invention, or a transfected or transformed cell of the invention.The invention also provides plant products, e.g., oils, seeds, leaves,extracts and the like, comprising a nucleic acid and/or a polypeptide ofthe invention. The transgenic plant can be dicotyledonous (a dicot) ormonocotyledonous (a monocot). The invention also provides methods ofmaking and using these transgenic plants and seeds. The transgenic plantor plant cell expressing a polypeptide of the present invention may beconstructed in accordance with any method known in the art. See, forexample, U.S. Pat. No. 6,309,872.

The recombinant expression, or over-expression, of the phytase moleculesof the invention may be achieved in combination with one or moreadditional molecules such as, for example, other enzymes. This approachis useful for producing combination products, such as a plant or plantpart that contains the instant phytase molecules as well as one or moreadditional molecules. The phytase molecules of this invention and theadditional molecules can be used in a combination treatment. Theresulting recombinantly expressed molecules may be used in homogenizedand/or purified form or alternatively in relatively unpurified form(e.g. as consumable plant parts that are useful when admixed with otherfoodstuffs for catalyzing the degradation of phytate).

In a particular aspect, the present invention provides for theexpression of phytase in transgenic plants or plant organs and methodsfor the production thereof. DNA expression constructs are provided forthe transformation of plants with a gene encoding phytase under thecontrol of regulatory sequences which are capable of directing theexpression of phytase. These regulatory sequences include sequencescapable of directing transcription in plants, either constitutively, orin stage and/or tissue specific manners.

The manner of expression depends, in part, on the use of the plant orparts thereof. The transgenic plants and plant organs provided by thepresent invention may be applied to a variety of industrial processeseither directly, e.g. in animal feeds or alternatively, the expressedphytase may be extracted and if desired, purified before application.Alternatively, the recombinant host plant or plant part may be useddirectly. In a particular aspect, the present invention provides methodsof catalyzing phytate-hydrolyzing reactions using seeds containingenhanced amounts of phytase. The method involves contacting transgenic,non-wild type seeds, e.g., in a ground or chewed form, withphytate-containing substrate and allowing the enzymes in the seeds toincrease the rate of reaction. By directly adding the seeds to aphytate-containing substrate, the invention provides a solution to theexpensive and problematic process of extracting and purifying theenzyme. In one exemplification the present invention provides methods oftreatment whereby an organism lacking a sufficient supply of an enzymeis administered the enzyme in the form of seeds containing enhancedamounts of the enzyme. In one aspect, the timing of the administrationof the enzyme to an organism is coordinated with the consumption of aphytate-containing foodstuff.

The expression of phytase in plants can be achieved by a variety ofmeans. Specifically, for example, technologies are available fortransforming a large number of plant species, including dicotyledonousspecies (e.g. tobacco, potato, tomato, Petunia, Brassica) and monocotspecies. Additionally, for example, strategies for the expression offoreign genes in plants are available. Additionally still, regulatorysequences from plant genes have been identified that are serviceable forthe construction of chimeric genes that can be functionally expressed inplants and in plant cells (e.g. Klee (1987) Ann. Rev. of Plant Phys.38:467-486; Clark et al. (1990) Virology December; 179(2):640-7; Smithet al. (1990) Mol. Gen. Genet. December; 224(3):477-81.

The introduction of gene constructs into plants can be achieved usingseveral technologies including transformation with Agrobacteriumtumefaciens or Agrobacterium rhizogenes. Non-limiting examples of planttissues that can be transformed thusly include protoplasts, microsporesor pollen, and explants such as leaves, stems, roots, hypocotyls, andcotyls. Furthermore, DNA can be introduced directly into protoplasts andplant cells or tissues by microinjection, electroporation, particlebombardment, and direct DNA uptake.

Proteins may be produced in plants by a variety of expression systems.For instance, the use of a constitutive promoter such as the 35Spromoter of Cauliflower Mosaic Virus (Guilley et al., 1982) isserviceable for the accumulation of the expressed protein in virtuallyall organs of the transgenic plant. Alternatively, the use of promotersthat are highly tissue-specific and/or stage-specific are serviceablefor this invention (Higgins, 1984; Shotwell, 1989) in order to biasexpression towards desired tissues and/or towards a desired stage ofdevelopment. The invention also uses protocols for expression in plantsof phytase molecules of the instant invention as disclosed in, forexample, U.S. Pat. No. 5,770,413 (Van Ooijen et al.) and U.S. Pat. No.5,593,963 (Van Ooijen et al.), that teaches use of fungal phytases.

Modification of Coding Sequences and Adjacent Sequences

The transgenic expression in plants of genes derived from heterologoussources may involve the modification of those genes to achieve andoptimize their expression in plants. In particular, bacterial ORFs whichencode separate enzymes but which are encoded by the same transcript inthe native microbe are best expressed in plants on separate transcripts.Thus, in one aspect, to achieve this, each microbial ORF is isolatedindividually and cloned within a cassette which provides a plantpromoter sequence at the 5′ end of the ORF and a plant transcriptionalterminator at the 3′ end of the ORF. The isolated ORF sequence canincludes the initiating ATG codon and the terminating STOP codon but mayinclude additional sequence beyond the initiating ATG and the STOPcodon. In addition, the ORF may be truncated, but still retain therequired activity; for particularly long ORFs, truncated versions whichretain activity may be preferable for expression in transgenicorganisms. “Plant promoters” and “plant transcriptional terminators”that can be used to practice this invention include any promoters and/ortranscriptional terminators which operate within plant cells. Thisincludes promoters and transcription terminators which may be derivedfrom non-plant sources such as viruses (an example is the CauliflowerMosaic Virus).

In some cases, modification to the ORF coding sequences and adjacentsequence is not required. It is sufficient to isolate a fragmentcontaining the ORF of interest and to insert it downstream of a plantpromoter. For example, Gaffney et. al. (Science 261: 754-756 (1993))have expressed the Pseudomonas nahG gene in transgenic plants under thecontrol of the CaMV 35S promoter and the CaMV tml terminatorsuccessfully without modification of the coding sequence and withnucleotides of the Pseudomonas gene upstream of the ATG still attached,and nucleotides downstream of the STOP codon still attached to the nahGORF. Preferably as little adjacent microbial sequence should be leftattached upstream of the ATG and downstream of the STOP codon. Inpractice, such construction may depend on the availability ofrestriction sites.

In other cases, the expression of genes derived from microbial sourcesmay provide problems in expression. These problems have been wellcharacterized in the art and are particularly common with genes derivedfrom certain microbial sources. These problems may apply to thenucleotide sequence of this invention and the modification of thesegenes can be undertaken using techniques now well known in the art. Thefollowing problems may be encountered:

Codon Usage

The invention provides nucleic acids having codons modified for usage inplants; in some cases preferred codon usage in plants differs from thepreferred codon usage in certain microorganisms. Comparison of the usageof codons within a cloned microbial ORF to usage in plant genes (and inparticular genes from the target plant) will enable an identification ofthe codons within the ORF which should preferably be changed. Typicallyplant evolution has tended towards a strong preference of thenucleotides C and G in the third base position of monocotyledons,whereas dicotyledons often use the nucleotides A or T at this position.By modifying a gene to incorporate preferred codon usage for aparticular target transgenic species, many of the problems describedbelow for GC/AT content and illegitimate splicing will be overcome.

GC/AT Content

The invention provides nucleic acids having their GC content modified,e.g., for usage in plants; plant genes typically have a GC content ofmore than 35%. ORF sequences which are rich in A and T nucleotides cancause several problems in plants. Firstly, motifs of ATTTA are believedto cause destabilization of messages and are found at the 3′ end of manyshort-lived mRNAs. Secondly, the occurrence of polyadenylation signalssuch as AATAAA at inappropriate positions within the message is believedto cause premature truncation of transcription. In addition,monocotyledons may recognize AT-rich sequences as splice sites (seebelow).

Sequences Adjacent to the Initiating Methionine

The invention provides nucleic acids having nucleotides adjacent to theATG modified and/or added; plants differ from microorganisms in thattheir messages do not possess a defined ribosome binding site. Rather,it is believed that ribosomes attach to the 5′ end of the message andscan for the first available ATG at which to start translation.Nevertheless, it is believed that there is a preference for certainnucleotides adjacent to the ATG and that expression of microbial genescan be enhanced by the inclusion of a eukaryotic consensus translationinitiator at the ATG. Clontech (1993/1994 catalog, page 210,incorporated herein by reference) have suggested one sequence as aconsensus translation initiator for the expression of the E. coli uidAgene in plants. Further, Joshi (N.A.R. 15: 6643-6653 (1987),incorporated herein by reference) has compared many plant sequencesadjacent to the ATG and suggests another consensus sequence. Insituations where difficulties are encountered in the expression ofmicrobial ORFs in plants, inclusion of one of these sequences at theinitiating ATG may improve translation. In such cases the last threenucleotides of the consensus may not be appropriate for inclusion in themodified sequence due to their modification of the second AA residue. Insome aspects, preferred sequences adjacent to the initiating methioninemay differ between different plant species. A survey of 14 maize geneslocated in the GenBank database provided the following results:

Position Before the Initiating ATG in 14 Maize Genes: −10 −9 −8 −7 −6 −5−4 −3 −2 −1 C 3 8 4 6 2 5 6 0 10 7 T 3 0 3 4 3 2 1 1 1 0 A 2 3 1 4 3 2 37 2 3 G 6 3 6 0 6 5 4 6 1 5This analysis can be done for the desired plant species into which thenucleotide sequence is being incorporated, and the sequence adjacent tothe ATG modified to incorporate the preferred nucleotides.Removal of Illegitimate Splice Sites

The invention provides nucleic acids having illegitimate splice sitesmodified or removed or functionally “knocked out”; genes cloned fromnon-plant sources and not optimized for expression in plants may alsocontain motifs which may be recognized in plants as 5′ or 3′ splicesites, and be cleaved, thus generating truncated or deleted messages.These sites can be removed using the techniques well known in the art.

Techniques for the modification of coding sequences and adjacentsequences are well known in the art. In cases where the initialexpression of a microbial ORF is low and it is deemed appropriate tomake alterations to the sequence as described above, then theconstruction of synthetic genes can be accomplished according to methodswell known in the art. These are, for example, described in thepublished patent disclosures EP 0 385 962 (to Monsanto), EP 0 359 472(to Lubrizol) and WO 93/07278 (to Ciba-Geigy), all of which areincorporated herein by reference. In most cases it is preferable toassay the expression of gene constructions using transient assayprotocols (which are well known in the art) prior to their transfer totransgenic plants.

Plant Promoters

The compositions of the invention may contain nucleic acid sequences,e.g., promoters, e.g., for transformation and expression in a plant ofinterest. The nucleic acid sequences may be present in DNA constructs orexpression cassettes. Nucleic acids of the invention can be, orcomprise, “expression cassettes”, including any nucleic acid moleculecapable of directing expression of a particular nucleotide sequence inan appropriate host cell comprising a promoter operatively linked to thenucleotide sequence of interest which is operatively linked totermination signals.

The compositions (e.g., nucleic acid sequences) of the invention alsocan comprise sequences required for proper translation of the nucleotidesequence. The coding region usually codes for a protein of interest butmay also code for a functional RNA of interest, for example antisenseRNA or a nontranslated RNA, in the sense or antisense direction. Theexpression cassette comprising the nucleotide sequence of interest maybe chimeric, meaning that at least one of its components is heterologouswith respect to at least one of its other components. The expressioncassette may also be one that is naturally occurring but has beenobtained in a recombinant form useful for heterologous expression.Typically, however, the expression cassette is heterologous with respectto the host, i.e., the particular DNA sequence of the expressioncassette does not occur naturally in the host cell and must have beenintroduced into the host cell or an ancestor of the host cell by atransformation event. The expression of the nucleotide sequence in theexpression cassette may be under the control of a constitutive promoteror of an inducible promoter that initiates transcription only when thehost cell is exposed to some particular external stimulus. Additionally,the promoter can also be specific to a particular tissue or organ orstage of development.

The present invention encompasses the transformation of plants withexpression cassettes capable of expressing polynucleotides. Theexpression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter) and a polynucleotide of interest. The expressioncassette may optionally comprise a transcriptional and translationaltermination region (i.e. termination region) functional in plants. Insome embodiments, the expression cassette comprises a selectable markergene to allow for selection for stable transformants. Expressionconstructs of the invention may also comprise a leader sequence and/or asequence allowing for inducible expression of the polynucleotide ofinterest. See, Guo et. al. (2003) Plant J. 34:383-92 and Chen et. al.(2003) Plant J. 36:731-40 for examples of sequences allowing forinducible expression.

The regulatory sequences of the expression construct are operably linkedto the polynucleotide of interest. By “operably linked” is intended afunctional linkage between a promoter and a second sequence wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleotide sequences being linked are contiguous.

Any promoter capable of driving expression in the plant of interest maybe used in the practice of the invention. The promoter may be native oranalogous or foreign or heterologous to the plant host. The terms“heterologous” and “exogenous” when used herein to refer to a nucleicacid sequence (e.g. a DNA or RNA sequence) or a gene, refer to asequence that originates from a source foreign to the particular hostcell or, if from the same source, is modified from its original form.Thus, a heterologous gene in a host cell includes a gene that isendogenous to the particular host cell but has been modified. The termsalso include non-naturally occurring multiple copies of a naturallyoccurring DNA sequence. Thus, the terms refer to a DNA segment that isforeign or heterologous to the cell, or homologous to the cell but in aposition within the host cell nucleic acid in which the element is notordinarily found. Exogenous DNA segments are expressed to yieldexogenous polypeptides. In alternative embodiments, a “homologous”nucleic acid (e.g. DNA) sequence is a nucleic acid (e.g. DNA or RNA)sequence naturally associated with a host cell into which it isintroduced.

The choice of promoters to be included depends upon several factors,including, but not limited to, efficiency, selectability, inducibility,desired expression level, and cell- or tissue-preferential expression.It is a routine matter for one of skill in the art to modulate theexpression of a sequence by appropriately selecting and positioningpromoters and other regulatory regions relative to that sequence.

Some suitable promoters initiate transcription only, or predominantly,in certain cell types. Thus, as used herein a cell type- ortissue-preferential promoter is one that drives expressionpreferentially in the target tissue, but may also lead to someexpression in other cell types or tissues as well. Methods foridentifying and characterizing promoter regions in plant genomic DNAinclude, for example, those described in the following references:Jordano, et. al., Plant Cell, 1:855-866 (1989); Bustos, et. al., PlantCell, 1:839-854 (1989); Green, et. al., EMBO J. 7, 4035-4044 (1988);Meier, et. al., Plant Cell, 3, 309-316 (1991); and Zhang, et. al., PlantPhysiology 110: 1069-1079 (1996).

Several tissue preferred regulated genes and/or promoters have beenreported in plants. Some reported tissue preferred genes include thegenes encoding the seed storage proteins (such as napin, cruciferin,beta-conglycinin, and phaseolin, prolamines, glutelins, globulins, andzeins) zeins or oil body proteins (such as oleosin), or genes involvedin fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACPdesaturase, and fatty acid desaturases (fad 2-1)), and other genesexpressed during embryo development (such as Bce4, see, for example, EP255378 and Kridl et. al., (1991) Seed Science Research, 1:209).

Examples of tissue-specific promoters, which have been described,include the lectin (Vodkin, Prog. Clin. Biol. Res., 138; 87 (1983);Lindstrom et. al., (1990) Der. Genet., 11:160), corn alcoholdehydrogenase 1 (Dennis et. al., Nucleic Acids Res., 12:3983 (1984)),corn light harvesting complex (see, e.g., Simpson, (1986) Science,233:34; Bansal (1992) Proc. Natl. Acad. Sci. USA 89:3654), corn heatshock protein (see, e.g., Odell et. al., (1985) Nature, 313:810; peasmall subunit RuBP carboxylase (see, e.g., Poulsen et. al., (1986) Mol.Gen. Genet., 205:193-200; Cashmore et. al., (1983) Gen. Eng. of Plants,Plenum Press, New York, 29-38); Ti plasmid mannopine synthase (see,e.g., Langridge et. al., (1989) Proc. Natl. Acad. Sci. USA,86:3219-3223), Ti plasmid nopaline synthase (Langridge et. al., (1989)Proc. Natl. Acad. Sci. USA, 86:3219-3223), petunia chalcone isomerase(see, e.g., vanTunen (1988) EMBO J. 7:1257); bean glycine rich protein 1(see, e.g., Keller (1989) Genes Dev. 3:1639); truncated CaMV 35s (see,e.g., Odell (1985) Nature 313:810); potato patatin (see, e.g., Wenzler(1989) Plant Mol. Biol. 13:347; root cell (see, e.g., Yamamoto (1990)Nucleic Acids Res. 18:7449); maize zein (see, e.g., Reina (1990) NucleicAcids Res. 18:6425; Lopes et. al. (1995) Mol. Gen. Genet. 247: 603-613;Kriz (1987) Mol. Gen. Genet. 207:90; Wandelt (1989) Nucleic Acids Res.,17:2354; Langridge (1983) Cell, 34:1015; Reina (1990) Nucleic AcidsRes., 18:7449), ADP-gpp promoter (see, e.g., U.S. Pat. No. 7,102,057);globulin-1 (see, e.g., Belanger (1991) Genetics 129:863); α-globulin(Sunilkumar, et. al. (2002), Transgenic Res. 11:347-359); α-tubulin; cab(see, e.g., Sullivan (1989) Mol. Gen. Genet., 215:431); PEPCase (seee.g., Hudspeth & Grula, (1989) Plant Molec. Biol., 12:579-589); R genecomplex-associated promoters (Chandler et. al., (1989) Plant Cell,1:1175); pea vicilin promoter (Czako et. al., (1992) Mol. Gen. Genet.,235:33; U.S. Pat. No. 5,625,136); GTL1 promoter (Takaiwa et. al. (1991)Plant Mol. Biol. 16 (1), 49-58); chalcone synthase promoters (Frankenet. al., (1991) EMBO J., 10:2605); GY1 promoter (Sims & Goldburg (1989)Nuc. Acid Res. 17(11) 4368) and the like; all of which are hereinincorporated by reference.

The invention can use fruit-preferred promoters, including any class offruit-preferred promoters, e.g., as expressed at or during antithesisthrough fruit development, at least until the beginning of ripening,e.g., as discussed in U.S. Pat. No. 4,943,674, the disclosure of whichis hereby incorporated by reference. The promoter for polygalacturonasegene is active in fruit ripening. The invention can use thepolygalacturonase gene as described, e.g., in U.S. Pat. No. 4,535,060,U.S. Pat. No. 4,769,061, U.S. Pat. No. 4,801,590, and U.S. Pat. No.5,107,065, which disclosures are incorporated herein by reference.

The invention can use any tissue-preferred promoters, including thosethat direct expression in leaf cells following damage to the leaf (forexample, from chewing insects), in tubers (for example, patatin genepromoter), and in fiber cells (an example of a developmentally-regulatedfiber cell protein is E6 (John & Crow (1992) PNAS 89:5769-5773). The E6gene is most active in fiber, although low levels of transcripts arefound in leaf, ovule and flower.

The invention can use promoters active in photosynthetic tissue, e.g.,in order to drive transcription in green tissues such as leaves andstems, are suitable when they drive expression only or predominantly insuch tissues. Alternatively, the invention can use promoters to conferexpression constitutively throughout the plant, or differentially withrespect to the green tissues, or differentially with respect to thedevelopmental stage of the green tissue in which expression occurs, orin response to external stimuli.

Exemplary promoters used to practice this invention include theribulose-1,5-bisphosphate carboxylase (RbcS) promoters such as the RbcSpromoter from eastern larch (Larix laricina), the pine cab6 promoter(Yamamoto et. al. (1994) Plant Cell Physiol. 35:773-778), the Cab-1 genepromoter from wheat (Fejes et. al. (1990) Plant Mol. Biol. 15:921-932),the CAB-1 promoter from spinach (Lubberstedt et. al. (1994) PlantPhysiol. 104:997-1006), the cab1R promoter from rice (Luan et. al.(1992) Plant Cell 4:971-981), the pyruvate orthophosphate dikinase(PPDK) promoter from corn (Matsuoka et. al. (1993) Proc Natl Acad SciUSA 90:9586-9590), the tobacco Lhcb1*2 promoter (Cerdan et. al. (1997)Plant Mol. Biol. 33:245-255), the Arabidopsis thaliana SUC2 sucrose-H+symporter promoter (Truernit et. al. (1995) Planta 196:564-570), andthylakoid membrane protein promoters from spinach (psaD, psaF, psaE, PC,FNR, atpC, atpD, cab, rbcS. Other promoters that drive transcription instems, leafs and green tissue are described in U.S. Patent PublicationNo. 2007/0006346, herein incorporated by reference in its entirety.

In some embodiments, the tissue specificity of some “tissue preferred”promoters may not be absolute and may be tested reporter genes such asGus or green fluorescent protein, cyan fluorescent protein, yellowfluorescent protein or red fluorescent protein. One can also achievetissue preferred expression with “leaky” expression by a combination ofdifferent tissue-preferred promoters. Other tissue preferred promoterscan be isolated by one skilled in the art (see U.S. Pat. No. 5,589,379).

In one aspect, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the nucleic acids ofthe invention. For example, the invention can use the auxin-responseelements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.)(Liu (1997) Plant Physiol. 115:397-407); the auxin-responsiveArabidopsis GST6 promoter (also responsive to salicylic acid andhydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); theauxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); aplant biotin response element (Streit (1997) Mol. Plant MicrobeInteract. 10:933-937); and, the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900-1902).

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents whichcan be applied to the plant, such as herbicides or antibiotic. Forexample, gene expression systems that are activated in the presence of achemical ligand, including ethanol, such as can be found in WO 96/27673;WO 93/01294; WO 94/03619; WO 02/061102, all of which are herebyincorporated by reference. The maize In2-2 promoter, activated bybenzenesulfonamide herbicide safeners, can be used (De Veylder (1997)Plant Cell Physiol. 38:568-577); application of different herbicidesafeners induces distinct gene expression patterns, including expressionin the root, hydathodes, and the shoot apical meristem. Coding sequencecan be under the control of, e.g., a tetracycline-inducible promoter,e.g., as described with transgenic tobacco plants containing the Avenasativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J.11:465-473); estrogen, such as, the ecdysone receptor (WO 01/52620) or,a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324). Using chemically- (e.g., hormone- or pesticide-) inducedpromoters, i.e., promoter responsive to a chemical which can be appliedto the transgenic plant in the field, expression of a polypeptide of theinvention can be induced at a particular stage of development of theplant.

Exemplary constitutive promoters which can be used to practice thisinvention, and which have been described, include rice actin 1 (Wang et.al. (1992) Mol. Cell. Biol., 12:3399; U.S. Pat. No. 5,641,876); otheractin isoforms (McElroy et. al. (1990) Plant Cell 2: 163-171 and McElroyet. al. (1991) Mol. Gen. Genet. 231: 150-160); CaMV 35S (Odell et. al.(1985) Nature, 313:810); CaMV 19S (Lawton et. al. (1987) Plant Mol.Biol. 9:315-324; U.S. Pat. No. 5,639,949); nos (Ebert et. al. (1987)PNAS USA 84:5745-5749); Adh (Walker et. al. (1987) PNAS USA84:6624-6628), sucrose synthase (Yang & Russell (1990) PNAS USA87:4144-4148); and the ubiquitin promoters (e.g. sunflower—Binet et. al.(1991) Plant Science 79: 87-94; maize—Christensen et. al. (1989) PlantMolec. Biol. 12: 619-632; and Arabidopsis—Callis et. al., J. Biol. Chem.(1990) 265:12486-12493; and Norris et. al., Plant Mol. Biol. (1993)21:895-906.

Any transcriptional terminator can be used to practice this invention,e.g., can be used in vectors, expression cassettes and the like. Theseare responsible for the termination of transcription beyond thetransgene and correct mRNA polyadenylation. The termination region maybe native with the transcriptional initiation region, may be native withthe operably linked DNA sequence of interest, may be native with theplant host, or may be derived from another source (i.e., foreign orheterologous to the promoter, the DNA sequence of interest, the planthost, or any combination thereof). Appropriate transcriptionalterminators are those that are known to function in plants and includethe CAMV 35S terminator, the tml terminator, the nopaline synthaseterminator and the pea rbcs E9 terminator. These can be used in bothmonocotyledons and dicotyledons. In addition, a gene's nativetranscription terminator may be used.

The invention can use any sequence to enhance gene expression fromwithin the transcriptional unit; and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants. For example, various intron sequenceshave been shown to enhance expression, particularly in monocotyledonouscells. For example, the introns of the maize Adhl gene have been foundto significantly enhance the expression of the wild-type gene under itscognate promoter when introduced into maize cells.

A number of non-translated leader sequences derived from viruses arealso known to enhance expression, and these are particularly effectivein dicotyledonous cells. Specifically, leader sequences from TobaccoMosaic Virus (TMV, the “W-sequence”), Maize Chlorotic Mottle Virus(MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effectivein enhancing expression (e.g. Gallie et. al. Nucl. Acids Res. 15:8693-8711 (1987); Skuzeski et. al. Plant Molec. Biol. 15: 65-79 (1990)).

Targeting of the Gene Product Within the Cell

Any mechanism for targeting gene products, e.g., in plants, can be usedto practice this invention, and such mechanisms are known to exist inplants and the sequences controlling the functioning of these mechanismshave been characterized in some detail. Sequences have beencharacterized which cause the targeting of gene products to other cellcompartments. Amino terminal sequences can be responsible for targetinga protein of interest to any cell compartment, such as, a vacuole,mitochondrion, peroxisome, protein bodies, endoplasmic reticulum,chloroplast, starch granule, amyloplast, apoplast or cell wall of aplant (e.g. Unger et. al. Plant Molec. Biol. 13: 411-418 (1989); Rogerset. al. (1985) Proc. Natl. Acad. Sci. USA 82: 6512-651; U.S. Pat. No.7,102,057; WO 2005/096704, all of which are hereby incorporated byreference). Optionally, the signal sequence may be an N-terminal signalsequence from waxy, an N-terminal signal sequence from γ-zein, a starchbinding domain, a C-terminal starch binding domain, a chloroplasttargeting sequence, which imports the mature protein to the chloroplast(Comai et. al. (1988) J. Biol. Chem. 263: 15104-15109; van den Broeck,et. al. (1985) Nature 313: 358-363; U.S. Pat. No. 5,639,949) or asecretion signal sequence from aleurone cells (Koehler & Ho, Plant Cell2: 769-783 (1990)). Additionally, amino terminal sequences inconjunction with carboxy terminal sequences are responsible for vacuolartargeting of gene products (Shinshi et. al. (1990) Plant Molec. Biol.14: 357-368).

In one aspect, the signal sequence selected should include the knowncleavage site, and the fusion constructed should take into account anyamino acids after the cleavage site(s), which are required for cleavage.In some cases this requirement may be fulfilled by the addition of asmall number of amino acids between the cleavage site and the transgeneATG or, alternatively, replacement of some amino acids within thetransgene sequence. These construction techniques are well known in theart and are equally applicable to any cellular compartment.

In one aspect, the above-described mechanisms for cellular targeting canbe utilized not only in conjunction with their cognate promoters, butalso in conjunction with heterologous promoters so as to effect aspecific cell-targeting goal under the transcriptional regulation of apromoter that has an expression pattern different to that of thepromoter from which the targeting signal derives.

In sum, a variety of means can be used to practice this invention,including any means to achieve the recombinant expression of phytase ina transgenic plant, seed, organ or any plant part. Such a transgenicplants and plant parts are serviceable as sources of recombinantlyexpressed phytase, which can be added directly to phytate-containingsources. Alternatively, the recombinant plant-expressed phytase can beextracted away from the plant source and, if desired, purified prior tocontacting the phytase substrate.

Within the context of the present invention, plants that can be selected(used to practice this invention) include, but are not limited to cropsproducing edible flowers such as cauliflower (Brassica oleracea),artichoke (Cynara scolymus), fruits such as apple (Malus, e.g.domesticus), banana (Musa, e.g. acuminata), berries (such as thecurrant, Ribes, e.g. rubrum), cherries (such as the sweet cherry,Prunus, e.g. avium), cucumber (Cucumis, e.g. sativus), grape (Vitis,e.g. vinifera), lemon (Citrus limon), melon (Cucumis melo), nuts (suchas the walnut, Juglans, e.g. regia; peanut, Arachis hypogeae), orange(Citrus, e.g. maxima), peach (Prunus, e.g. persica), pear (Pyra, e.g.communis), plum (Prunus, e.g. domestics), strawberry (Fragaria, e.g.moschata), tomato (Lycopersicon, e.g. esculentum), leafs, such asalfalfa (Medicago, e.g. sativa), cabbages (e.g. Brassica oleracea),endive (Cichoreum, e.g. endivia), leek (Allium, e.g. porrum), lettuce(Lactuca, e.g. sativa), spinach (Spinacia, e.g. oleraceae), tobacco(Nicotiana, e.g. tabacum), roots, such as arrowroot (Maranta, e.g.arundinacea), beet (Beta, e.g. vulgaris), carrot (Daucus, e.g. carota),cassava (Manihot, e.g. esculenta), turnip (Brassica, e.g. rapa), radish(Raphanus, e.g. sativus), yam (Dioscorea, e.g. esculenta), sweet potato(Ipomoea batatas) and seeds, such as bean (Phaseolus, e.g. vulgaris),pea (Pisum, e.g. sativum), soybean (Glycin, e.g. max), wheat (Triticum,e.g. aestivum), barley (Hordeum, e.g. vulgare), corn (Zea, e.g. mays),rice (Oryza, e.g. sativa), rapeseed (Brassica napus), millet (PanicumL.), sunflower (Helianthus annus), oats (Avena sativa), tubers, such askohlrabi (Brassica, e.g. oleraceae), potato (Solanum, e.g. tuberosum)and the like.

In one aspect, the nucleic acids and polypeptides of the invention areexpressed in or inserted in any plant or seed. Transgenic plants of theinvention can be dicotyledonous or monocotyledonous. Examples of monocottransgenic plants of the invention are grasses, such as meadow grass(blue grass, Poa), forage grass such as festuca, lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn). Examples of dicot transgenic plants ofthe invention are tobacco, legumes, such as lupins, potato, sugar beet,pea, bean and soybean, and cruciferous plants (family Brassicaceae),such as cauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Thus, the transgenic plants and seeds of theinvention include a broad range of plants, including, but not limitedto, species from the genera Anacardium, Arachis, Asparagus, Atropa,Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana,Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum,Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

In alternative embodiments, the nucleic acids of the invention areexpressed in plants which contain fiber cells, including, e.g., cotton,silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush,winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca andflax. In alternative embodiments, the transgenic plants of the inventioncan be members of the genus Gossypium, including members of anyGossypium species, such as G. arboreum; G. herbaceum, G. barbadense, andG. hirsutum.

Additional plants as well as non-plant expression systems can be used topractice this invention. The choice of the plant species is primarilydetermined by the intended use of the plant or parts thereof and theamenability of the plant species to transformation.

Several techniques are available for the introduction of the expressionconstruct containing the phytase-encoding DNA sequence into the targetplants. Techniques for transforming a wide variety of higher plantspecies are well known and described in the technical and scientificliterature. See, e.g., Weising (1988) Ann. Rev. Genet. 22:421-477; U.S.Pat. No. 5,750,870. Such techniques also can include but are not limitedto transformation of protoplasts using the calcium/polyethylene glycolmethod, electroporation and microinjection or (coated) particlebombardment (Potrykus, 1990). In addition to these so-called direct DNAtransformation methods, transformation systems involving vectors arewidely available, such as viral vectors (e.g. from the CauliflowerMosaic Cirus (CaMV) and bacterial vectors (e.g. from the genusAgrobacterium) (Potrykus, 1990). After selection and/or screening, theprotoplasts, cells or plant parts that have been transformed can beregenerated into whole plants, using methods known in the art (Horsch etal., 1985). The choice of the transformation and/or regenerationtechniques is not critical for this invention.

Nucleic acids and expression constructs of the invention can beintroduced into a plant cell by any means. In alternative aspects ofpracticing this invention, the term “introducing” in the context of apolynucleotide, for example, a nucleotide construct of interest, isintended to mean presenting to the plant the polynucleotide in such amanner that the polynucleotide gains access to the interior of a cell ofthe plant. Where more than one polynucleotide is to be introduced, thesepolynucleotides can be assembled as part of a single nucleotideconstruct, or as separate nucleotide constructs, and can be located onthe same or different transformation vectors. Accordingly, thesepolynucleotides can be introduced into the host cell of interest in asingle transformation event, in separate transformation events, or, forexample, in plants, as part of a breeding protocol. The methods of theinvention do not depend on a particular method for introducing one ormore polynucleotides into a plant, only that the polynucleotide(s) gainsaccess to the interior of at least one cell of the plant. Methods forintroducing polynucleotides into plants are known in the art including,but not limited to, transient transformation methods, stabletransformation methods, and virus-mediated methods.

“Transient transformation” can be used to practice this invention, andin some aspects in the context of a polynucleotide is intended to meanthat a polynucleotide is introduced into the plant and does notintegrate into the genome of the plant. “Stably introducing” or “stablyintroduced” in the context of a polynucleotide introduced into a plantcan be used to practice this invention, and in some aspects it isintended that the introduced polynucleotide is stably incorporated intothe plant genome, and thus the plant is stably transformed with thepolynucleotide.

“Stable transformation” or “stably transformed” in the context of apolynucleotide introduced into a plant can be used to practice thisinvention, and in some aspects it is intended that a polynucleotide, forexample, a nucleotide construct described herein, is introduced into aplant integrates into the genome of the plant and is capable of beinginherited by the progeny thereof, more particularly, by the progeny ofmultiple successive generations. Introduction into the genome of adesired plant can be such that the enzyme is regulated by endogenoustranscriptional or translational control elements. Transformationtechniques for both monocotyledons and dicotyledons are well known inthe art.

The nucleic acids of the invention can be used to confer desired traitson essentially any plant. In one embodiment, the enzyme of the inventionmay be expressed in such a way that the enzyme will not come in contactwith it's substrate until desired. For example, an enzyme of theinvention may be targeted and retained in the endoplasmic reticulum of aplant cell. Retention of the enzyme, in the endoplasmic reticulum of thecell, will prevent the enzyme from coming in contact with its substrate.The enzyme and substrate may then be brought into contact through anymeans able to disrupt the subcellular architecture, such as, grinding,milling, heating, and the like. See, WO 98/11235, WO 2003/18766, and WO2005/096704, all of which are hereby incorporated by reference.

Selectable marker genes can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide.Selection markers used routinely in transformation include the nptllgene, which confers resistance to kanamycin and related antibiotics(Messing & Vierra. Gene 19: 259-268 (1982); Bevan et. al., Nature304:184-187 (1983)), the bar gene, which confers resistance to theherbicide phosphinothricin (White et. al., Nucl. Acids Res 18: 1062(1990), Spencer et. al. Theor. Appl. Genet. 79: 625-631 (1990)), the hphgene, which confers resistance to the antibiotic hygromycin (Blochinger& Diggelmann, Mol Cell Biol 4: 2929-2931), the dhfr gene, which confersresistance to methotrexate (Bourouis et. al., EMBO J. 2(7): 1099-1104(1983)), the EPSPS gene, which confers resistance to glyphosate (U.S.Pat. Nos. 4,940,935 and 5,188,642),

Alternatively, transgenic plant material can be identified through apositive selection system, such as, the system utilizing themannose-6-phosphate isomerase gene, which provides the ability tometabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629).

In one aspect, making transgenic plants or seeds comprises incorporatingsequences of the invention and, optionally, marker genes into a targetexpression construct (e.g., a plasmid), along with positioning of thepromoter and the terminator sequences. This can involve transferring themodified gene into the plant through a suitable method. One or more ofthe sequences of the invention may be combined with sequences thatconfer resistance to insect, disease, drought, increase yield, improvenutritional quality of the grain, improve ethanol yield and the like.

For example, a construct may be introduced directly into the genomic DNAof the plant cell using techniques such as electroporation andmicroinjection of plant cell protoplasts, or the constructs can beintroduced directly to plant tissue using ballistic methods, such as DNAparticle bombardment. For example, see, e.g., Christou (1997) Plant Mol.Biol. 35:197-203; Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein(1987) Nature 327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69,discussing use of particle bombardment to introduce transgenes intowheat; and Adam (1997) supra, for use of particle bombardment tointroduce YACs into plant cells. For example, Rinehart (1997) supra,used particle bombardment to generate transgenic cotton plants.Apparatus for accelerating particles is described U.S. Pat. No.5,015,580; and, the commercially available BioRad (Biolistics) PDS-2000particle acceleration instrument; see also, John, U.S. Pat. No.5,608,148; and Ellis, U.S. Pat. No. 5,681,730, describingparticle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with anucleic acids, e.g., an expression construct. Although plantregeneration from protoplasts is not easy with cereals, plantregeneration is possible in legumes using somatic embryogenesis fromprotoplast derived callus. Organized tissues can be transformed withnaked DNA using gene gun technique, where DNA is coated on tungstenmicroprojectiles, shot 1/100th the size of cells, which carry the DNAdeep into cells and organelles. Transformed tissue is then induced toregenerate, usually by somatic embryogenesis. This technique has beensuccessful in several cereal species including maize and rice.

Nucleic acids, e.g., expression constructs, can also be introduced in toplant cells using recombinant viruses. Plant cells can be transformedusing viral vectors, such as, e.g., tobacco mosaic virus derived vectors(Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use ofviral replicons for the expression of genes in plants,” Mol. Biotechnol.5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl.Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springerlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids of the invention, including important cereals,see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch,Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci. USA80:4803; Thykjaer (1997) supra; Park (1996) Plant Mol. Biol.32:1135-1148, discussing T-DNA integration into genomic DNA. See alsoD'Halluin, U.S. Pat. No. 5,712,135, describing a process for the stableintegration of a DNA comprising a gene that is functional in a cell of acereal, or other monocotyledonous plant.

In one aspect, the third step can involve selection and regeneration ofwhole plants capable of transmitting the incorporated target gene to thenext generation. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker that has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee(1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants fromtransgenic tissues such as immature embryos, they can be grown undercontrolled environmental conditions in a series of media containingnutrients and hormones, a process known as tissue culture. Once wholeplants are generated and produce seed, evaluation of the progeny begins.

In one aspect, after the expression cassette is stably incorporated intransgenic plants, it can be introduced into other plants by sexualcrossing. Any of a number of standard breeding techniques can be used,depending upon the species to be crossed. See, for example, Welsh J. R.,Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY(1981); Crop Breeding, Wood D. R. (Ed.) American Society of AgronomyMadison, Wis. (1983); Mayo O., The Theory of Plant Breeding, SecondEdition, Clarendon Press, Oxford (1987); Singh, D. P., Breeding forResistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); andWricke and Weber, Quantitative Genetics and Selection Plant Breeding,Walter de Gruyter and Co., Berlin (1986).

In one aspect, since transgenic expression of the nucleic acids of theinvention leads to phenotypic changes, plants comprising the recombinantnucleic acids of the invention can be sexually crossed with a secondplant to obtain a final product. Thus, the seed of the invention can bederived from a cross between two transgenic plants of the invention, ora cross between a plant of the invention and another plant. The desiredeffects (e.g., expression of the polypeptides of the invention toproduce a plant in which flowering behavior is altered) can be enhancedwhen both parental plants express the polypeptides (e.g., phytase) ofthe invention. The desired effects can be passed to future plantgenerations by standard propagation means.

For dicots, a binary vector system can be used (Hoekema et al., 1983; EP0120516 Schilperoort et al.). For example, Agrobacterium strains can beused which contain a vir plasmid with the virulence genes and acompatible plasmid containing the gene construct to be transferred. Thisvector can replicate in both E. coli and in Agrobacterium, and isderived from the binary vector Bin19 (Bevan, 1984) that is altered indetails that are not relevant for this invention. The binary vectors asused in this example contain between the left- and right-bordersequences of the T-DNA, an identical NPTII-gene coding for kanamycinresistance (Bevan, 1984) and a multiple cloning site to clone in therequired gene constructs.

The transformation and regeneration of monocotyledonous crops can bepracticed using any method or standard procedure; monocots are amenableto transformation and fertile transgenic plants can be regenerated fromtransformed cells. In an alternative aspect, monocots are transformed byAgrobacterium transformation.

In one aspect, transgenic rice plants can be obtained using thebacterial hph gene, encoding hygromycin resistance, as a selectionmarker; the gene can be introduced by electroporation. In one aspect,transgenic maize plants can be obtained by introducing the Streptomyceshygroscopicus bar gene, which encodes phosphinothricin acetyltransferase(an enzyme which inactivates the herbicide phosphinothricin), intoembryogenic cells of a maize suspension culture by microparticlebombardment. In one aspect, genetic material can be introduced intoaleurone protoplasts of monocot crops such as wheat and barley. In oneaspect, wheat plants are regenerated from embryogenic suspension cultureby selecting only the aged compact and nodular embryogenic callustissues for the establishment of the embryogenic suspension cultures. Inone aspect, the combination with transformation systems for these cropsenables the application of the present invention to monocots. Thesemethods and other methods may also be applied for the transformation andregeneration of dicots.

In practicing this invention, expression of the phytase constructinvolves such details as transcription of the gene by plant polymerases,translation of mRNA, etc. that are known to persons skilled in the artof recombinant DNA techniques. Some details relevant for the practicingsome embodiments of this invention are discussed herein. Regulatorysequences which are known or are found to cause expression of phytasemay be used in the present invention. The choice of the regulatorysequences used can depend on the target crop and/or target organ ofinterest. Such regulatory sequences may be obtained from plants or plantviruses, or may be chemically synthesized. Such regulatory sequences arepromoters active in directing transcription in plants, eitherconstitutively or stage and/or tissue specific, depending on the use ofthe plant or parts thereof. These promoters include, but are not limitedto promoters showing constitutive expression, such as the 35S promoterof Cauliflower Mosaic Virus (CaMV) (Guilley et al., 1982), those forleaf-specific expression, such as the promoter of the ribulosebisphosphate carboxylase small subunit gene (Coruzzi et al., 1984),those for root-specific expression, such as the promoter from theglutamine synthase gene (Tingey et al., 1987), those for seed-specificexpression, such as the cruciferin A promoter from Brassica napus (Ryanet al., 1989), those for tuber-specific expression, such as the class-Ipatatin promoter from potato (Koster-Topfer et al., 1989; Wenzler etal., 1989) or those for fruit-specific expression, such as thepolygalacturonase (PG) promoter from tomato (Bird et al., 1988).

Other regulatory sequences such as terminator sequences andpolyadenylation signals can be used to practice this invention, and theycan include any such sequence functioning as such in plants, the choiceof which is within the level of the skilled artisan. An example of suchsequences is the 3′ flanking region of the nopaline synthase (nos) geneof Agrobacterium tumefaciens (Bevan, supra). The regulatory sequencesmay also include enhancer sequences, such as found in the 35S promoterof CaMV, and mRNA stabilizing sequences such as the leader sequence ofAlfalfa Mosaic Cirus (AlMV) RNA4 (Brederode et al., 1980) or any othersequences functioning in a like manner.

In some embodiments, a phytase of the invention is expressed in anenvironment that allows for stability of the expressed protein. Thechoice of cellular compartments, such as cytosol, endoplasmic reticulum,vacuole, protein body or periplasmic space can be used in the presentinvention to create such a stable environment, depending on thebiophysical parameters of the phytase. Such parameters include, but arenot limited to pH-optimum, sensitivity to proteases or sensitivity tothe molarity of the preferred compartment.

In some embodiments, a phytase of the invention is expressed incytoplasm; in some aspect, to obtain expression in the cytoplasm of thecell, the expressed enzyme should not contain a secretory signal peptideor any other target sequence. For expression in chloroplasts andmitochondria the expressed enzyme should contain specific so-calledtransit peptide for import into these organelles. Targeting sequencesthat can be attached to the enzyme of interest in order to achieve thisare known (Smeekens et al., 1990; van den Broeck et al., 1985; Wolter etal., 1988). If the activity of the enzyme is desired in the vacuoles asecretory signal peptide has to be present, as well as a specifictargeting sequence that directs the enzyme to these vacuoles (Tague etal., 1990). The same is true for the protein bodies in seeds. The DNAsequence encoding the enzyme of interest should be modified in such away that the enzyme can exert its action at the desired location in thecell.

In some embodiments, to achieve extracellular expression of the phytase,the expression construct of the present invention utilizes a secretorysignal sequence. Although signal sequences which are homologous (native)to the plant host species may be preferred, heterologous signalsequences, i.e. those originating from other plant species or ofmicrobial origin, may be used as well. Such signal sequences are knownto those skilled in the art. Appropriate signal sequences which may beused within the context of the present invention are disclosed in Blobelet al., 1979; Von Heijne, 1986; Garcia et al., 1987; Sijmons et al.,1990; Ng et al., 1994; and Powers et al., 1996).

In some embodiments, all parts of the relevant DNA constructs(promoters, regulatory-, secretory-, stabilizing-, targeting-, ortermination sequences) of the present invention are modified, ifdesired, to affect their control characteristics using methods known tothose skilled in the art. Plants containing phytase obtained via thepresent invention may be used to obtain plants or plant organs with yethigher phytase levels. For example, it may be possible to obtain suchplants or plant organs by the use of somoclonal variation techniques orby cross breeding techniques. Such techniques are well known to thoseskilled in the art.

In one aspect, the instant invention provides a method (and productsthereof) of achieving a highly efficient overexpression system forphytase and other molecules. In one aspect, the invention provides amethod (and products thereof) of achieving a highly efficientoverexpression system for phytase and pH 2.5 acid phosphatase inTrichoderma. This system results in enzyme compositions that haveparticular utility in the animal feed industry. Additional detailsregarding this approach are in the public literature and/or are known tothe skilled artisan. In a particular non-limiting exemplification, suchpublicly available literature includes EP 0659215 (WO 9403612 A1)(Nevalainen et al.), although these reference do not teach the inventivemolecules of the instant application.

In some embodiments, the invention uses in vivo reassortment, which canbe focused on “inter-molecular” processes collectively referred to as“recombination”, which in bacteria can be a “RecA-dependent” phenomenon.The invention can rely on recombination processes of a host cell torecombine and re-assort sequences, or the cells' ability to mediatereductive processes to decrease the complexity of quasi-repeatedsequences in the cell by deletion. This process of “reductivereassortment” occurs by an “intra-molecular”, RecA-independent process.

Therefore, in another aspect of the invention, variant polynucleotidescan be generated by the process of reductive reassortment. The methodinvolves the generation of constructs containing consecutive sequences(original encoding sequences), their insertion into an appropriatevector, and their subsequent introduction into an appropriate host cell.The reassortment of the individual molecular identities occurs bycombinatorial processes between the consecutive sequences in theconstruct possessing regions of homology, or between quasi-repeatedunits. The reassortment process recombines and/or reduces the complexityand extent of the repeated sequences, and results in the production ofnovel molecular species. Various treatments may be applied to enhancethe rate of reassortment. These could include treatment withultra-violet light, or DNA damaging chemicals, and/or the use of hostcell lines displaying enhanced levels of “genetic instability”. Thus thereassortment process may involve homologous recombination or the naturalproperty of quasi-repeated sequences to direct their own evolution.

The invention can use repeated or “quasi-repeated” sequences; thesesequence can play a role in genetic instability. In the presentinvention, “quasi-repeats” are repeats that are not restricted to theiroriginal unit structure. Quasi-repeated units can be presented as anarray of sequences in a construct; consecutive units of similarsequences. Once ligated, the junctions between the consecutive sequencesbecome essentially invisible and the quasi-repetitive nature of theresulting construct is now continuous at the molecular level. Thedeletion process the cell performs to reduce the complexity of theresulting construct operates between the quasi-repeated sequences. Thequasi-repeated units provide a practically limitless repertoire oftemplates upon which slippage events can occur. The constructscontaining the quasi-repeats thus effectively provide sufficientmolecular elasticity that deletion (and potentially insertion) eventscan occur virtually anywhere within the quasi-repetitive units.

In some aspects, when the quasi-repeated sequences are all ligated inthe same orientation, for instance head to tail or vice versa, the cellcannot distinguish individual units. Consequently, the reductive processcan occur throughout the sequences. In contrast, when for example, theunits are presented head to head, rather than head to tail, theinversion delineates the endpoints of the adjacent unit so that deletionformation will favor the loss of discrete units. Thus, in one aspect ofthe invention the sequences are in the same orientation. Randomorientation of quasi-repeated sequences will result in the loss ofreassortment efficiency, while consistent orientation of the sequenceswill offer the highest efficiency. However, while having fewer of thecontiguous sequences in the same orientation decreases the efficiency,it can still provide sufficient elasticity for the effective recovery ofnovel molecules. Constructs can be made with the quasi-repeatedsequences in the same orientation to allow higher efficiency.

Sequences can be assembled in a head to tail orientation using any of avariety of methods, including the following:

-   -   (a) Primers that include a poly-A head and poly-T tail which        when made single-stranded provide orientation can be utilized.        This is accomplished by having the first few bases of the        primers made from RNA and hence easily removed RNAse H.    -   (b) Primers that include unique restriction cleavage sites can        be utilized. Multiple sites, a battery of unique sequences, and        repeated synthesis and ligation steps would be required.    -   (c) The inner few bases of the primer can be thiolated and an        exonuclease used to produce properly tailed molecules.

In some aspects, the recovery of the re-assorted sequences relies on theidentification of cloning vectors with a reduced RI. The re-assortedencoding sequences can then be recovered by amplification. The productsare re-cloned and expressed. The recovery of cloning vectors withreduced RI can be effected by:

-   -   1) The use of vectors only stably maintained when the construct        is reduced in complexity;    -   2) The physical recovery of shortened vectors by physical        procedures. In this case, the cloning vector is recovered using        standard plasmid isolation procedures and size fractionated on        either an agarose gel, or column with a low molecular weight cut        off utilizing standard procedures;    -   3) The recovery of vectors containing interrupted genes which        can be selected when insert size decreases; and    -   4) The use of direct selection techniques with an expression        vector and the appropriate selection.

Encoding sequences (for example, genes) from related organisms can beused to practice this invention, and they can demonstrate a high degreeof homology and encode quite diverse protein products. These types ofsequences are particularly useful in the present invention asquasi-repeats. However, while the exemplary protocols discussed belowdemonstrate the reassortment of nearly identical original encodingsequences (quasi-repeats), this process is not limited to such nearlyidentical repeats.

Once formed, the constructs may or may not be size fractionated on anagarose gel according to published protocols, inserted into a cloningvector, and transfected into an appropriate host cell. The cells arethen propagated and “reductive reassortment” is effected. The rate ofthe reductive reassortment process may be stimulated by the introductionof DNA damage if desired. Whether the reduction in RI is mediated bydeletion formation between repeated sequences by an “intra-molecular”mechanism, or mediated by recombination-like events through“inter-molecular” mechanisms is immaterial. The end result is areassortment of the molecules into all possible combinations.

In one aspect, methods of this invention comprise the additional step ofscreening the library members of the shuffled pool to identifyindividual shuffled library members having the ability to bind orotherwise interact, or catalyze a particular reaction (e.g., such ascatalyzing the hydrolysis of a phytate).

In one aspect, the polypeptides that are identified from such librariescan be used for therapeutic, diagnostic, research and related purposes(e.g., catalysts, solutes for increasing osmolarity of an aqueoussolution, and the like), and/or can be subjected to one or moreadditional cycles of shuffling and/or selection.

In another aspect, prior to or during recombination or reassortment,polynucleotides of the invention or polynucleotides generated by themethod described herein can be subjected to agents or processes whichpromote the introduction of mutations into the original polynucleotides.The introduction of such mutations would increase the diversity ofresulting hybrid polynucleotides and polypeptides encoded therefrom. Theagents or processes which promote mutagenesis can include, but are notlimited to: (+)-CC-1065, or a synthetic analog such as(+)-CC-1065-(N-3-Adenine, see Sun and Hurley, 1992); an N-acetylated ordeacetylated 4′-fluoro-4-aminobiphenyl adduct capable of inhibiting DNAsynthesis (see, for example, van de Poll et al., 1992); or aN-acetylated or deacetylated 4-aminobiphenyl adduct capable ofinhibiting DNA synthesis (see also, van de Poll et al., 1992, pp.751-758); trivalent chromium, a trivalent chromium salt, a polycyclicaromatic hydrocarbon (“PAH”) DNA adduct capable of inhibiting DNAreplication, such as 7-bromomethyl-benz[a]anthracene (“BMA”),tris(2,3-dibromopropyl)phosphate (“Tris-BP”),1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA),benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II)halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline(“N-hydroxy-IQ”), andN-hydroxy-2-amino-1-methyl-6-phenylmidazo[4,5-f]-pyridine(“N-hydroxy-PhIP”). An exemplary means for slowing or halting PCRamplification consist of UV light (+)-CC-1065 and(+)-CC-1065-(N-3-Adenine). Particularly encompassed means are DNAadducts or polynucleotides comprising the DNA adducts from thepolynucleotides or polynucleotides pool, which can be released orremoved by a process including heating the solution comprising thepolynucleotides prior to further processing.

In another aspect, the invention is directed to a method of producingrecombinant proteins having biological activity by treating a samplecomprising double-stranded template polynucleotides encoding a wild typeprotein under conditions according to the invention which provide forthe production of hybrid or re-assorted polynucleotides.

The invention also provides for the use of proprietary codon primers(containing a degenerate N,N,G/T sequence) to introduce point mutationsinto a polynucleotide, so as to generate a set of progeny polypeptidesin which a full range of single amino acid substitutions is representedat each amino acid position (gene site saturated mutagenesis (GSSM)).The oligos used are comprised contiguously of a first homologoussequence, a degenerate N,N,G/T sequence, and optionally a secondhomologous sequence. The downstream progeny translational products fromthe use of such oligos include all possible amino acid changes at eachamino acid site along the polypeptide, because the degeneracy of theN,N,G/T sequence includes codons for all 20 amino acids.

In one aspect, one such degenerate oligo (comprised of one degenerateN,N,G/T cassette) is used for subjecting each original codon in aparental polynucleotide template to a full range of codon substitutions.In another aspect, at least two degenerate N,N,G/T cassettes areused—either in the same oligo or not, for subjecting at least twooriginal codons in a parental polynucleotide template to a full range ofcodon substitutions. Thus, more than one N,N,G/T sequence can becontained in one oligo to introduce amino acid mutations at more thanone site. This plurality of N,N,G/T sequences can be directlycontiguous, or separated by one or more additional nucleotidesequence(s). In another aspect, oligos serviceable for introducingadditions and deletions can be used either alone or in combination withthe codons containing an N,N,G/T sequence, to introduce any combinationor permutation of amino acid additions, deletions, and/or substitutions.

In one aspect, it is possible to simultaneously mutagenize two or morecontiguous amino acid positions using an oligo that contains contiguousN,N,G/T triplets, i.e. a degenerate (N,N,G/T)_(n) sequence.

In another aspect, the present invention provides for the use ofdegenerate cassettes having less degeneracy than the N,N,G/T sequence.For example, it may be desirable in some instances to use (e.g. in anoligo) a degenerate triplet sequence comprised of only one N, where saidN can be in the first second or third position of the triplet. Any otherbases including any combinations and permutations thereof can be used inthe remaining two positions of the triplet. Alternatively, it may bedesirable in some instances to use (e.g., in an oligo) a degenerateN,N,N triplet sequence, or an N,N, G/C triplet sequence.

It is appreciated, however, that the use of a degenerate triplet (suchas N,N,G/T or an N,N, G/C triplet sequence) as disclosed in the instantinvention is advantageous for several reasons. In one aspect, thisinvention provides a means to systematically and fairly easily generatethe substitution of the full range of possible amino acids (for a totalof 20 amino acids) into each and every amino acid position in apolypeptide. Thus, for a 100 amino acid polypeptide, the inventionprovides a way to systematically and fairly easily generate 2000distinct species (i.e., 20 possible amino acids per position times 100amino acid positions). It is appreciated that there is provided, throughthe use of an oligo containing a degenerate N,N,G/T or an N,N, G/Ctriplet sequence, 32 individual sequences that code for 20 possibleamino acids. Thus, in a reaction vessel in which a parentalpolynucleotide sequence is subjected to saturation mutagenesis using onesuch oligo, there are generated 32 distinct progeny polynucleotidesencoding 20 distinct polypeptides. In contrast, the use of anon-degenerate oligo in site-directed mutagenesis leads to only oneprogeny polypeptide product per reaction vessel.

This invention also provides for the use of nondegenerate oligos, whichcan optionally be used in combination with degenerate primers disclosed.It is appreciated that in some situations, it is advantageous to usenondegenerate oligos to generate specific point mutations in a workingpolynucleotide. This provides a means to generate specific silent pointmutations, point mutations leading to corresponding amino acid changes,and point mutations that cause the generation of stop codons and thecorresponding expression of polypeptide fragments.

Thus, in one aspect, each saturation mutagenesis reaction vesselcontains polynucleotides encoding at least 20 progeny polypeptidemolecules such that all 20 amino acids are represented at the onespecific amino acid position corresponding to the codon positionmutagenized in the parental polynucleotide. The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g., cloned into asuitable E. coli host using an expression vector) and subjected toexpression screening. When an individual progeny polypeptide isidentified by screening to display a favorable change in property (whencompared to the parental polypeptide), it can be sequenced to identifythe correspondingly favorable amino acid substitution contained therein.

It is appreciated that upon mutagenizing each and every amino acidposition in a parental polypeptide using gene site saturationmutagenesis (GSSM) as disclosed herein, favorable amino acid changes maybe identified at more than one amino acid position. One or more newprogeny molecules can be generated that contain a combination of all orpart of these favorable amino acid substitutions. For example, if 2specific favorable amino acid changes are identified in each of 3 aminoacid positions in a polypeptide, the permutations include 3possibilities at each position (no change from the original amino acid,and each of two favorable changes) and 3 positions. Thus, there are3×3×3 or 27 total possibilities, including 7 that were previouslyexamined—6 single point mutations (i.e., 2 at each of three positions)and no change at any position.

In yet another aspect, site-saturation mutagenesis can be used togetherwith shuffling, chimerization, recombination and other mutagenizingprocesses, along with screening. This invention provides for the use ofany mutagenizing process(es), including saturation mutagenesis, in aniterative manner. In one exemplification, the iterative use of anymutagenizing process(es) is used in combination with screening.

Thus, in a non-limiting exemplification, polynucleotides andpolypeptides of the invention can be derived by gene site saturationmutagenesis (GSSM) in combination with additional mutagenizationprocesses, such as process where two or more related polynucleotides areintroduced into a suitable host cell such that a hybrid polynucleotideis generated by recombination and reductive reassortment.

In addition to performing mutagenesis along the entire sequence of agene, mutagenesis can be used to replace each of any number of bases ina polynucleotide sequence, wherein the number of bases to be mutagenizedcan be every integer from 15 to 100,000. Thus, instead of mutagenizingevery position along a molecule, one can subject every or a discretenumber of bases (can be a subset totaling from 15 to 100,000) tomutagenesis. A separate nucleotide can be used for mutagenizing eachposition or group of positions along a polynucleotide sequence. A groupof 3 positions to be mutagenized may be a codon. The mutations can beintroduced using a mutagenic primer, containing a heterologous cassette,also referred to as a mutagenic cassette. Exemplary cassettes can havefrom 1 to 500 bases. Each nucleotide position in such heterologouscassettes be N, A, C, G, T, A/C, A/G, A/T, C/G, C/T, G/T, C/G/T, A/G/T,A/C/T, A/C/G, or E, where E is any base that is not A, C, G, or T (E canbe referred to as a designer oligo).

In a general sense, saturation mutagenesis comprises mutagenizing acomplete set of mutagenic cassettes (wherein each cassette can be about1-500 bases in length) in defined polynucleotide sequence to bemutagenized (wherein the sequence to be mutagenized can be from about 15to 100,000 bases in length). Thus, a group of mutations (ranging from 1to 100 mutations) is introduced into each cassette to be mutagenized. Agrouping of mutations to be introduced into one cassette can bedifferent or the same from a second grouping of mutations to beintroduced into a second cassette during the application of one round ofsaturation mutagenesis. Such groupings are exemplified by deletions,additions, groupings of particular codons, and groupings of particularnucleotide cassettes.

Defined sequences to be mutagenized include a whole gene, pathway, cDNA,an entire open reading frame (ORF), and entire promoter, enhancer,repressor/transactivator, origin of replication, intron, operator, orany polynucleotide functional group. Generally, a “defined sequences”for this purpose may be any polynucleotide that a 15 base-polynucleotidesequence, and polynucleotide sequences of lengths between 15 bases and15,000 bases (this invention specifically names every integer inbetween). Considerations in choosing groupings of codons include typesof amino acids encoded by a degenerate mutagenic cassette.

In one aspect, a grouping of mutations that can be introduced into amutagenic cassette, this invention specifically provides for degeneratecodon substitutions (using degenerate oligos) that code for 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acidsat each position, and a library of polypeptides encoded thereby.

In alternative aspects nucleic acids of the invention comprise DNA,including cDNA, genomic DNA, and synthetic DNA. The DNA may bedouble-stranded or single-stranded, and if single stranded may be thecoding strand or non-coding (anti-sense) strand. Alternatively, thenucleic acids of the invention may comprise RNA.

As discussed in more detail below, the isolated nucleic acid sequencesof the invention may be used to prepare the polypeptides of theinvention.

Accordingly, another aspect of the invention is an isolated nucleic acidsequence which encodes a polypeptide of the invention. A nucleic acidsequence of the invention can comprise additional coding sequences, suchas leader sequences or proprotein sequences and non-coding sequences,such as introns or non-coding sequences 5′ and/or 3′ of the codingsequence. Thus, as used herein, the term “polynucleotide encoding apolypeptide” encompasses a polynucleotide which includes only codingsequence for the polypeptide as well as a polynucleotide which includesadditional coding and/or non-coding sequence.

Alternatively, the nucleic acid sequences of the invention may bemutagenized using conventional techniques, such as site directedmutagenesis, or other techniques familiar to those skilled in the art,to introduce silent changes into the polynucleotide of the invention. Asused herein, “silent changes” include, for example, changes that do notalter the amino acid sequence encoded by the polynucleotide. Suchchanges may be desirable in order to increase the level of thepolypeptide produced by host cells containing a vector encoding thepolypeptide by introducing codons or codon pairs that occur frequentlyin the host organism.

The invention also relates to polynucleotides that have nucleotidechanges which result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptides of the invention. Suchnucleotide changes may be introduced using techniques such as sitedirected mutagenesis, random chemical mutagenesis, exonuclease IIIdeletion, and other recombinant DNA techniques.

Where necessary, conditions which permit the probe to specificallyhybridize to complementary sequences may be determined by placing theprobe in contact with complementary sequences from samples known tocontain the complementary sequence as well as control sequences which donot contain the complementary sequence. Hybridization conditions, suchas the salt concentration of the hybridization buffer, the formamideconcentration of the hybridization buffer, or the hybridizationtemperature, may be varied to identify conditions which allow the probeto hybridize specifically to complementary nucleic acids.

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product.

Many methods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures, and dot blots. Protocols for each of theseprocedures are provided in Ausubel et al. Current Protocols in MolecularBiology, John Wiley 503 Sons, Inc. 1997 and Sambrook et al., MolecularCloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor LaboratoryPress, 1989.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). Typically, the probes compriseoligonucleotides. In one aspect, the amplification reaction may comprisea PCR reaction. PCR protocols are described in Ausubel and Sambrook,supra. Alternatively, the amplification may comprise a ligase chainreaction, 3SR, or strand displacement reaction. (See Barany, F., “TheLigase Chain Reaction in a PCR World,” PCR Methods and Applications1:5-16, 1991; E. Fahy et al., “Self-sustained Sequence Replication(3SR): An Isothermal Transcription-based Amplification SystemAlternative to PCR”, PCR Methods and Applications 1:25-33, 1991; andWalker G. T. et al., “Strand Displacement Amplification—an Isothermal invitro DNA Amplification Technique”, Nucleic Acid Research 20:1691-1696,1992). In such procedures, the nucleic acids in the sample are contactedwith the probes, the amplification reaction is performed, and anyresulting amplification product is detected. The amplification productmay be detected by performing gel electrophoresis on the reactionproducts and staining the gel with an intercalator such as ethidiumbromide. Alternatively, one or more of the probes may be labeled with aradioactive isotope and the presence of a radioactive amplificationproduct may be detected by autoradiography after gel electrophoresis.

Probes derived from sequences near the ends of a sequence of theinvention may also be used in chromosome walking procedures to identifyclones containing genomic sequences located adjacent to the nucleic acidsequences as set forth above. Such methods allow the isolation of geneswhich encode additional proteins from the host organism.

A nucleic acid sequence of the invention can be used as a probe toidentify and isolate related nucleic acids. In some aspects, the relatednucleic acids may be cDNAs or genomic DNAs from organisms other than theone from which the nucleic acid was isolated. For example, the otherorganisms may be related organisms. In such procedures, a nucleic acidsample is contacted with the probe under conditions which permit theprobe to specifically hybridize to related sequences. Hybridization ofthe probe to nucleic acids from the related organism is then detectedusing any of the methods described above.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

Hybridization may be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10×Denhardt's, and 0.5 mg/ml polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4−9×10⁸ cpm/ug) of ³²Pend-labeled oligonucleotide probe are then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh1×SET at Tm-10° C. for the oligonucleotide probe. The membrane is thenexposed to auto-radiographic film for detection of hybridizationsignals.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, T_(m), isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the T_(m) for a particular probe. The melting temperature of theprobe may be calculated using the following formulas: For probes between14 and 70 nucleotides in length the melting temperature (T_(m)) iscalculated using the formula: T_(m)=81.5+16.6(log [Na+])+0.41(fractionG+C)−(600/N), where N is the length of the probe. If the hybridizationis carried out in a solution containing formamide, the meltingtemperature may be calculated using the equation: T_(m)=81.5+16.6(log[Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N), where N is thelength of the probe. Prehybridization may be carried out in 6×SSC,5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured fragmented salmonsperm DNA or 6×SSC, 5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denaturedfragmented salmon sperm DNA, 50% formamide. The formulas for SSC andDenhardt's solutions can be found, e.g., in Sambrook et al., supra.

Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the Tm. For shorterprobes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the T_(m). Typically, for hybridizations in6×SSC, the hybridization is conducted at approximately 68° C. Usually,for hybridizations in 50% formamide containing solutions, thehybridization is conducted at approximately 42° C. All of the foregoinghybridizations are considered to be under conditions of high stringency.

Following hybridization, the filter is washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content), and the nucleic acid type (e.g., RNA v. DNA). Examplesof progressively higher stringency condition washes are as follows:2×SSC, 0.1% SDS at room temperature for 15 minutes (low stringency);0.1×SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderatestringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes at between thehybridization temperature and 68° C. (high stringency); and 0.15M NaClfor 15 minutes at 72° C. (very high stringency). A final low stringencywash can be conducted in 0.1×SSC at room temperature. The examples aboveare merely illustrative of one set of conditions that can be used towash filters. One of skill in the art would know that there are numerousrecipes for different stringency washes. Some other examples are givenbelow.

Nucleic acids which have hybridized to the probe can be identified byautoradiography or other conventional techniques.

The above procedure may be modified to identify nucleic acids havingdecreasing levels of homology to the probe sequence. For example, toobtain nucleic acids of decreasing homology to the detectable probe,less stringent conditions may be used. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a Na+ concentration of approximately1 M. Following hybridization, the filter may be washed with 2×SSC, 0.5%SDS at the temperature of hybridization. These conditions are consideredto be “moderate” conditions above 50° C. and “low” conditions below 50°C. A specific example of “moderate” hybridization conditions is when theabove hybridization is conducted at 55° C. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

For example, the preceding methods may be used to isolate nucleic acidshaving a sequence with at least about 99%, at least 98%, at least 97%,at least 95%, at least 90%, or at least 80% homology to a nucleic acidsequence as set forth in SEQ ID NO:1, sequences substantially identicalthereto, or fragments comprising at least about 10, 15, 20, 25, 30, 35,40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases thereof,and the sequences complementary to any of the foregoing sequences.Homology may be measured using an alignment algorithm. For example, thehomologous polynucleotides may have a coding sequence which is anaturally occurring allelic variant of one of the coding sequencesdescribed herein. Such allelic variants may have a substitution,deletion or addition of one or more nucleotides when compared to anucleic acid sequence as set forth in SEQ ID NO:1, or sequencescomplementary thereto.

Additionally, the above procedures may be used to isolate nucleic acidswhich encode polypeptides having at least about 99%, at least 95%, atleast 90%, at least 85%, at least 80%, or at least 70% homology to apolypeptide having a sequence as set forth in SEQ ID NO:2, sequencessubstantially identical thereto, or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof as determined using a sequence alignment algorithm (e.g., suchas the FASTA version 3.0t78 algorithm with the default parameters).

Another aspect of the invention is an isolated or purified polypeptidecomprising a sequence as set forth in SEQ ID NO:1, sequencessubstantially identical thereto, or fragments comprising at least about5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive aminoacids thereof. As discussed above, such polypeptides may be obtained byinserting a nucleic acid encoding the polypeptide into a vector suchthat the coding sequence is operably linked to a sequence capable ofdriving the expression of the encoded polypeptide in a suitable hostcell. For example, the expression vector may comprise a promoter, aribosome binding site for translation initiation and a transcriptionterminator. The vector may also include appropriate sequences foramplifying expression.

Promoters suitable for expressing the polypeptide or fragment thereof inbacteria include the E. coli lac or trp promoters, the lad promoter, thelacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter, thelambda P_(R) promoter, the lambda P_(L) promoter, promoters from operonsencoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), andthe acid phosphatase promoter. Fungal promoters include the ∀ factorpromoter. Eukaryotic promoters include the CMV immediate early promoter,the HSV thymidine kinase promoter, heat shock promoters, the early andlate SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter. Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses may also beused.

Mammalian expression vectors may also comprise an origin of replication,any necessary ribosome binding sites, a polyadenylation site, splicedonor and acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells may also contain enhancers to increase expression levels Enhancersare cis-acting elements of DNA, usually from about 10 to about 300 bp inlength that act on a promoter to increase its transcription. Examplesinclude the SV40 enhancer on the late side of the replication origin bp100 to 270, the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and the adenovirusenhancers.

In addition, the expression vectors typically contain one or moreselectable marker genes to permit selection of host cells containing thevector. Such selectable markers include genes encoding dihydrofolatereductase or genes conferring neomycin resistance for eukaryotic cellculture, genes conferring tetracycline or ampicillin resistance in E.coli, and the S. cerevisiae TRP1 gene.

The probe DNA used for selectively isolating the target DNA of interestfrom the DNA derived from at least one microorganism can be afull-length coding region sequence or a partial coding region sequenceof DNA for an enzyme of known activity. The original DNA library can beprobed using mixtures of probes comprising at least a portion of the DNAsequence encoding an enzyme having the specified enzyme activity. Theseprobes or probe libraries can be single-stranded and the microbial DNAwhich is probed can be converted into single-stranded form. The probesthat are suitable are those derived from DNA encoding enzymes having anactivity similar or identical to the specified enzyme activity which isto be screened.

The probe DNA can be at least about 10 bases or at least 15 bases. Inone aspect, the entire coding region may be employed as a probe.Conditions for the hybridization in which target DNA is selectivelyisolated by the use of at least one DNA probe will be designed toprovide a hybridization stringency of at least about 50% sequenceidentity, more particularly a stringency providing for a sequenceidentity of at least about 70%.

The probe DNA can be “labeled” with one partner of a specific bindingpair (i.e. a ligand) and the other partner of the pair is bound to asolid matrix to provide ease of separation of target from its source.The ligand and specific binding partner can be selected from, in eitherorientation, the following: (1) an antigen or hapten and an antibody orspecific binding fragment thereof; (2) biotin or iminobiotin and avidinor streptavidin; (3) a sugar and a lectin specific therefor; (4) anenzyme and an inhibitor therefor; (5) an apoenzyme and cofactor; (6)complementary homopolymeric oligonucleotides; and (7) a hormone and areceptor therefor. The solid phase can be selected from: (1) a glass orpolymeric surface; (2) a packed column of polymeric beads; and (3)magnetic or paramagnetic particles.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is ligated to thedesired position in the vector following digestion of the insert and thevector with appropriate restriction endonucleases. Alternatively, bluntends in both the insert and the vector may be ligated. A variety ofcloning techniques are disclosed in Ausubel et al. Current Protocols inMolecular Biology, John Wiley 503 Sons, Inc. 1997 and Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press, 1989. Such procedures and others are deemed to bewithin the scope of those skilled in the art.

The vector may be, for example, in the form of a plasmid, a viralparticle, or a phage. Other vectors include chromosomal, nonchromosomaland synthetic DNA sequences, derivatives of SV40; bacterial plasmids,phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, and pseudorabies. A variety of cloning andexpression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989).

Particular bacterial vectors which may be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

The host cell may be any of the host cells familiar to those skilled inthe art, including prokaryotic cells, eukaryotic cells, mammalian cells,insect cells, or plant cells. As representative examples of appropriatehosts, there may be mentioned: bacterial cells, such as E. coli,Streptomyces, Bacillus subtilis, Bacillus cereus, Salmonella typhimuriumand various species within the genera Pseudomonas, Streptomyces andStaphylococcus, fungal cells, such as Aspergillus, yeast such as anyspecies of Pichia, Saccharomyces, Schizosaccharomyces, Schwanniomyces,including Pichia pastoris, Saccharomyces cerevisiae, orSchizosaccharomyces pombe, insect cells such as Drosophila S2 andSpodoptera Sf9, animal cells such as CHO, COS or Bowes melanoma, andadenoviruses. The selection of an appropriate host is within theabilities of those skilled in the art.

The vector may be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract is retained forfurther purification. Microbial cells employed for expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents. Such methods are well known to those skilled in the art.The expressed polypeptide or fragment thereof can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the polypeptide. Ifdesired, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts (described by Gluzman,Cell, 23:175, 1981), and other cell lines capable of expressing proteinsfrom a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK celllines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.Additional details relating to the recombinant expression of proteinsare available to those skilled in the art. For example, ProteinExpression: A Practical Approach (Practical Approach Series by S. J.Higgins (Editor), B. D. Hames (Editor) (July 1999) Oxford UniversityPress; ISBN: 0199636249 provides ample guidance to the those skilled inthe art for the expression of proteins in a wide variety of organisms.

Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers. In other aspects,fragments or portions of the polypeptides may be employed for producingthe corresponding full-length polypeptide by peptide synthesis;therefore, the fragments may be employed as intermediates for producingthe full-length polypeptides.

As known by those skilled in the art, the nucleic acid sequences of theinvention can be optimized for expression in a variety of organisms. Inone aspect, sequences of the invention are optimized for codon usage inan organism of interest, e.g., a fungus such as S. cerevisiae or abacterium such as E. coli. Optimization of nucleic acid sequences forthe purpose of codon usage is well understood in the art to refer to theselection of a particular codon favored by an organism to encode aparticular amino acid. Optimized codon usage tables are known for manyorganisms. For example, see Transfer RNA in Protein Synthesis by DolphL. Hatfield, Byeong J. Lee, Robert M. Pirtle (Editor) (July 1992) CRCPress; ISBN: 0849356989. Thus, the invention also includes nucleic acidsof the invention adapted for codon usage of an organism.

Optimized expression of nucleic acid sequences of the invention alsorefers to directed or random mutagenesis of a nucleic acid to effectincreased expression of the encoded protein. The mutagenesis of thenucleic acids of the invention can directly or indirectly provide for anincreased yield of expressed protein. By way of non-limiting example,mutagenesis techniques described herein may be utilized to effectmutation of the 5′ untranslated region, 3′ untranslated region, orcoding region of a nucleic acid, the mutation of which can result inincreased stability at the RNA or protein level, thereby resulting in anincreased yield of protein.

Cell-free translation systems can also be employed to produce one of thepolypeptides of the invention, using mRNAs transcribed from a DNAconstruct comprising a promoter operably linked to a nucleic acidencoding the polypeptide or fragment thereof. In some aspects, the DNAconstruct may be linearized prior to conducting an in vitrotranscription reaction. The transcribed mRNA is then incubated with anappropriate cell-free translation extract, such as a rabbit reticulocyteextract, to produce the desired polypeptide or fragment thereof.

The invention also relates to variants of the polypeptides of theinvention. The term “variant” includes derivatives or analogs of thesepolypeptides. In particular, the variants may differ in amino acidsequence from the polypeptides of the invention, and sequencessubstantially identical thereto, by one or more substitutions,additions, deletions, fusions and truncations, which may be present inany combination.

The variants may be naturally occurring or created in vitro. Inparticular, such variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures.

Other methods of making variants are also familiar to those skilled inthe art. These include procedures in which nucleic acid sequencesobtained from natural isolates are modified to generate nucleic acidswhich encode polypeptides having characteristics which enhance theirvalue in industrial or laboratory applications. In such procedures, alarge number of variant sequences having one or more nucleotidedifferences with respect to the sequence obtained from the naturalisolate are generated and characterized. Typically, these nucleotidedifferences result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described in Leung, D. W., et al., Technique, 1:11-15, 1989) andCaldwell, R. C. and Joyce G. F., PCR Methods Applic., 2:28-33, 1992.Briefly, in such procedures, nucleic acids to be mutagenized are mixedwith PCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction may be performed using 20 fmoles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl₂, 0.5 mMMnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedthat these parameters may be varied as appropriate. The mutagenizednucleic acids are cloned into an appropriate vector and the activitiesof the polypeptides encoded by the mutagenized nucleic acids isevaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described in Reidhaar-Olson, J. F. andSauer, R. T., et al., Science, 241:53-57, 1988. Briefly, in suchprocedures a plurality of double stranded oligonucleotides bearing oneor more mutations to be introduced into the cloned DNA are synthesizedand inserted into the cloned DNA to be mutagenized. Clones containingthe mutagenized DNA are recovered and the activities of the polypeptidesthey encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in pending U.S. patentapplication Ser. No. 08/677,112 filed Jul. 9, 1996, entitled, Method of“DNA Shuffling with Polynucleotides Produced by Blocking or interruptinga Synthesis or Amplification Process”.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described inStemmer, W. P., PNAS, USA, 91:10747-10751, 1994. Briefly, in suchprocedures a plurality of nucleic acids to be recombined are digestedwith DNase to generate fragments having an average size of 50-200nucleotides. Fragments of the desired average size are purified andresuspended in a PCR mixture. PCR is conducted under conditions whichfacilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/:l in a solution of 0.2 mM of each dNTP, 2.2mM MgCl2, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5units of Taq polymerase per 100:1 of reaction mixture is added and PCRis performed using the following regime: 94° C. for 60 seconds, 94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45times) and 72° C. for 5 minutes. However, it will be appreciated thatthese parameters may be varied as appropriate. In some aspects,oligonucleotides may be included in the PCR reactions. In other aspects,the Klenow fragment of DNA polymerase I may be used in a first set ofPCR reactions and Taq polymerase may be used in a subsequent set of PCRreactions. Recombinant sequences are isolated and the activities of thepolypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In some aspects,random mutations in a sequence of interest are generated by propagatingthe sequence of interest in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described in PCTPublication No. WO 91/16427, published Oct. 31, 1991, entitled “Methodsfor Phenotype Creation from Multiple Gene Populations”.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described in Arkin, A. P. and Youvan, D. C., PNAS, USA,89:7811-7815, 1992.

In some aspects, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described inDelegrave, S, and Youvan, D. C., Biotechnol. Res., 11:1548-1552, 1993.Random and site-directed mutagenesis are described in Arnold, F. H.,Current Opinion in Biotechnology, 4:450-455, 1993.

In some aspects, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in pendingU.S. patent application Ser. No. 08/677,112 filed Jul. 9, 1996,entitled, “Method of DNA Shuffling with Polynucleotides Produced byBlocking or interrupting a Synthesis or Amplification Process”, andpending U.S. patent application Ser. No. 08/651,568 filed May 22, 1996,entitled, “Combinatorial Enzyme Development.”

The variants of the polypeptides of the invention may be variants inwhich one or more of the amino acid residues of the polypeptides of theinvention are substituted with a conserved or non-conserved amino acidresidue (e.g., a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code.

Conservative substitutions are those that substitute a given amino acidin a polypeptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the followingreplacements: replacements of an aliphatic amino acid such as Ala, Val,Leu and Ile with another aliphatic amino acid; replacement of a Ser witha Thr or vice versa; replacement of an acidic residue such as Asp andGlu with another acidic residue; replacement of a residue bearing anamide group, such as Asn and Gln, with another residue bearing an amidegroup; exchange of a basic residue such as Lys and Arg with anotherbasic residue; and replacement of an aromatic residue such as Phe, Tyrwith another aromatic residue.

Other variants are those in which one or more of the amino acid residuesof the polypeptides of the invention includes a substituent group.

Still other variants are those in which the polypeptide is associatedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol).

Additional variants are those in which additional amino acids are fusedto the polypeptide, such as a leader sequence, a secretory sequence, aproprotein sequence or a sequence which facilitates purification,enrichment, or stabilization of the polypeptide. In some aspects,derivatives and analogs retain the same biological function or activityas the polypeptides of the invention, and can include a proprotein, suchthat the fragment, derivative, or analog can be activated by cleavage ofthe proprotein portion to produce an active polypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying phytase-encoding nucleicacids to modify codon usage. In one aspect, the invention providesmethods for modifying codons in a nucleic acid encoding a phytase toincrease or decrease its expression in a host cell. The invention alsoprovides nucleic acids encoding a phytase modified to increase itsexpression in a host cell, phytase enzymes so modified, and methods ofmaking the modified phytase enzymes. The method comprises identifying a“non-preferred” or a “less preferred” codon in phytase-encoding nucleicacid and replacing one or more of these non-preferred or less preferredcodons with a “preferred codon” encoding the same amino acid as thereplaced codon and at least one non-preferred or less preferred codon inthe nucleic acid has been replaced by a preferred codon encoding thesame amino acid. A preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,insect cells and mammalian cells. Thus, the invention provides methodsfor optimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as Escherichiacoli and Pseudomonas fluorescens; gram positive bacteria, such asLactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris,Bacillus subtilis. Exemplary host cells also include eukaryoticorganisms, e.g., various yeast, such as Saccharomyces sp., includingSaccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris,and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, andmammalian cells and cell lines and insect cells and cell lines. Thus,the invention also includes nucleic acids and polypeptides optimized forexpression in these organisms and species.

For example, the codons of a nucleic acid encoding an phytase isolatedfrom a bacterial cell are modified such that the nucleic acid isoptimally expressed in a bacterial cell different from the bacteria fromwhich the phytase was derived, a yeast, a fungi, a plant cell, an insectcell or a mammalian cell. Methods for optimizing codons are well knownin the art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int. J.Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188;Narum (2001) Infect. Immun. 69:7250-7253. See also Narum (2001) Infect.Immun. 69:7250-7253, describing optimizing codons in mouse systems;Outchkourov (2002) Protein Expr. Purif. 24:18-24, describing optimizingcodons in yeast; Feng (2000) Biochemistry 39:15399-15409, describingoptimizing codons in E. coli; Humphreys (2000) Protein Expr. Purif20:252-264, describing optimizing codon usage that affects secretion inE. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide, an expression cassette or vector or a transfectedor transformed cell of the invention. The transgenic non-human animalscan be, e.g., goats, rabbits, sheep, pigs, cows, rats and mice,comprising the nucleic acids of the invention. These animals can beused, e.g., as in vivo models to study phytase activity, or, as modelsto screen for modulators of phytase activity in vivo. The codingsequences for the polypeptides to be expressed in the transgenicnon-human animals can be designed to be constitutive, or, under thecontrol of tissue-specific, developmental-specific or inducibletranscriptional regulatory factors. Transgenic non-human animals can bedesigned and generated using any method known in the art; see, e.g.,U.S. Pat. Nos. 6,211,428; 6,187,992; 6,156,952; 6,118,044; 6,111,166;6,107,541; 5,959,171; 5,922,854; 5,892,070; 5,880,327; 5,891,698;5,639,940; 5,573,933; 5,387,742; 5,087,571, describing making and usingtransformed cells and eggs and transgenic mice, rats, rabbits, sheep,pigs and cows. See also, e.g., Pollock (1999) J. Immunol. Methods231:147-157, describing the production of recombinant proteins in themilk of transgenic dairy animals; Baguisi (1999) Nat. Biotechnol.17:456-461, demonstrating the production of transgenic goats. U.S. Pat.No. 6,211,428, describes making and using transgenic non-human mammalswhich express in their brains a nucleic acid construct comprising a DNAsequence. U.S. Pat. No. 5,387,742, describes injecting clonedrecombinant or synthetic DNA sequences into fertilized mouse eggs,implanting the injected eggs in pseudo-pregnant females, and growing toterm transgenic mice whose cells express proteins related to thepathology of Alzheimer's disease. U.S. Pat. No. 6,187,992, describesmaking and using a transgenic mouse whose genome comprises a disruptionof the gene encoding amyloid precursor protein (APP).

“Knockout animals” can also be used to practice the methods of theinvention. For example, in one aspect, the transgenic or modifiedanimals of the invention comprise a “knockout animal,” e.g., a “knockoutmouse,” engineered not to express or to be unable to express a phytase.

In another aspect, transgenic non-human organisms are provided whichcontain a heterologous sequence encoding a phytase of the invention(e.g., the specifically enumerated sequence modifications of SEQ IDNO:2). Various methods to make the transgenic animals of the subjectinvention can be employed. Generally speaking, three such methods may beemployed. In one such method, an embryo at the pronuclear stage (a “onecell embryo”) is harvested from a female and the transgene ismicroinjected into the embryo, in which case the transgene will bechromosomally integrated into both the germ cells and somatic cells ofthe resulting mature animal. In another such method, embryonic stemcells are isolated and the transgene incorporated therein byelectroporation, plasmid transfection or microinjection, followed byreintroduction of the stem cells into the embryo where they colonize andcontribute to the germ line. Methods for microinjection of mammalianspecies is described in U.S. Pat. No. 4,873,191.

In yet another exemplary method, embryonic cells are infected with aretrovirus containing the transgene whereby the germ cells of the embryohave the transgene chromosomally integrated therein. When the animals tobe made transgenic are avian, because avian fertilized ova generally gothrough cell division for the first twenty hours in the oviduct,microinjection into the pronucleus of the fertilized egg is problematicdue to the inaccessibility of the pronucleus. Therefore, of the methodsto make transgenic animals described generally above, retrovirusinfection is preferred for avian species, for example as described inU.S. Pat. No. 5,162,215. If micro-injection is to be used with avianspecies, however, a published procedure by Love et al., (Biotechnol., 12Jan. 1994) can be utilized whereby the embryo is obtained from asacrificed hen approximately two and one-half hours after the laying ofthe previous laid egg, the transgene is microinjected into the cytoplasmof the germinal disc and the embryo is cultured in a host shell untilmaturity. When the animals to be made transgenic are bovine or porcine,microinjection can be hampered by the opacity of the ova thereby makingthe nuclei difficult to identify by traditional differentialinterference-contrast microscopy. To overcome this problem, the ova canfirst be centrifuged to segregate the pronuclei for bettervisualization.

In one aspect, the “non-human animals” of the invention include bovine,porcine, ovine and avian animals (e.g., cow, pig, sheep, chicken). The“transgenic non-human animals” of the invention are produced byintroducing “transgenes” into the germline of the non-human animal.Embryonal target cells at various developmental stages can be used tointroduce transgenes. Different methods are used depending on the stageof development of the embryonal target cell. The zygote is the besttarget for micro-injection. The use of zygotes as is target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host gene before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene.

In one aspect, the term “transgenic” is used to describe an animal whichincludes exogenous genetic material within all of its cells. A“transgenic” animal can be produced by cross-breeding two chimericanimals which include exogenous genetic material within cells used inreproduction. Twenty-five percent of the resulting offspring will betransgenic i.e., animals which include the exogenous genetic materialwithin all of their cells in both alleles, 50% of the resulting animalswill include the exogenous genetic material within one allele and 25%will include no exogenous genetic material.

In one aspect, a microinjection method is used to practice theinvention. The transgene is digested and purified free from any vectorDNA, e.g., by gel electrophoresis. In one aspect, the transgene includesan operatively associated promoter which interacts with cellularproteins involved in transcription, ultimately resulting in constitutiveexpression. Promoters useful in this regard include those fromcytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes virus,as well as those from the genes encoding metallothionin, skeletal actin,P-enolpyruvate carboxylase (PEPCK), phosphoglycerate (PGK), DHFR, andthymidine kinase. Promoters for viral long terminal repeats (LTRs) suchas Rous Sarcoma Virus can also be employed. When the animals to be madetransgenic are avian, preferred promoters may include those for thechicken β-globin gene, chicken lysozyme gene, and avian leukosis virus.Constructs useful in plasmid transfection of embryonic stem cells willemploy additional regulatory elements well known in the art such asenhancer elements to stimulate transcription, splice acceptors,termination and polyadenylation signals, and ribosome binding sites topermit translation.

Retroviral infection can also be used to introduce transgene into anon-human animal, as described above. The developing non-human embryocan be cultured in vitro to the blastocyst stage. During this time, theblastomeres can be targets for retroviral infection (Jaenich, R., Proc.Natl. Acad. Sci. USA 73:1260-1264, 1976). Efficient infection of theblastomeres is obtained by enzymatic treatment to remove the zonapellucida (Hogan, et al. (1986) in Manipulating the Mouse Embryo, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The viralvector system used to introduce the transgene is typically areplication-defective retro virus carrying the transgene (Jahner, etal., Proc. Natl. Acad. Sci. USA 82: 6927-6931, 1985; Van der Putten, etal., Proc. Natl. Acad. Sci. USA 82: 6148-6152, 1985). Transfection iseasily and efficiently obtained by culturing the blastomeres on amonolayer of virus-producing cells (Van der Putten, supra; Stewart, etal., EMBO J. 6: 383-388, 1987). Alternatively, infection can beperformed at a later stage. Virus or virus-producing cells can beinjected into the blastocoele (D. Jahner et al., Nature 298: 623-628,1982). Most of the founders will be mosaic for the transgene sinceincorporation occurs only in a subset of the cells which formed thetransgenic nonhuman animal. Further, the founder may contain variousretro viral insertions of the transgene at different positions in thegenome which generally will segregate in the offspring. In addition, itis also possible to introduce transgenes into the germ line, albeit withlow efficiency, by intrauterine retroviral infection of the midgestationembryo (D. Jahner et al., supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (M. J. Evans et al., Nature292:154-156, 1981; M. O. Bradley et al., Nature 309:255-258, 1984;Gossler, et al., Proc. Natl. Acad. Sci. USA 83:9065-9069, 1986; andRobertson et al., Nature 322:445-448, 1986). Transgenes can beefficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter becombined with blastocysts from a nonhuman animal. The ES cellsthereafter colonize the embryo and contribute to the germ line of theresulting chimeric animal. (For review see Jaenisch, R., Science240:1468-1474, 1988).

In one aspect, the “transformed” means a cell into which (or into anancestor of which) has been introduced, by means of recombinant nucleicacid techniques, a heterologous nucleic acid molecule. “Heterologous”refers to a nucleic acid sequence that either originates from anotherspecies or is modified from either its original form or the formprimarily expressed in the cell.

In one aspect, the “transgene” means any piece of DNA which is insertedby artifice into a cell, and becomes part of the genome of the organism(i.e., either stably integrated or as a stable extrachromosomal element)which develops from that cell. Such a transgene may include a gene whichis partly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. Included within this definition is a transgene created bythe providing of an RNA sequence which is transcribed into DNA and thenincorporated into the genome. The transgenes of the invention includeDNA sequences which encode phytases or polypeptides having phytaseactivity, and include polynucleotides, which may be expressed in atransgenic non-human animal. The term “transgenic” as used hereinadditionally includes any organism whose genome has been altered by invitro manipulation of the early embryo or fertilized egg or by anytransgenic technology to induce a specific gene knockout. The term “geneknockout” as used herein, refers to the targeted disruption of a gene invivo with complete loss of function that has been achieved by anytransgenic technology familiar to those in the art. In one aspect,transgenic animals having gene knockouts are those in which the targetgene has been rendered nonfunctional by an insertion targeted to thegene to be rendered non-functional by homologous recombination.

In one aspect, the term “transgenic” includes any transgenic technologyfamiliar to those in the art which can produce an organism carrying anintroduced transgene or one in which an endogenous gene has beenrendered non-functional or “knocked out.”

The transgene to be used in the practice of the subject invention is aDNA sequence comprising a sequence coding for a phytase or a polypeptidehaving phytase activity. In one aspect, a polynucleotide having asequence as set forth in SEQ ID NO:1 or a sequence encoding apolypeptide having a sequence as set forth in SEQ ID NO:2 is thetransgene as the term is defined herein. Where appropriate, DNAsequences that encode proteins having phytase activity but differ innucleic acid sequence due to the degeneracy of the genetic code may alsobe used herein, as may truncated forms, allelic variants andinterspecies homologues.

In one aspect, after an embryo has been microinjected, colonized withtransfected embryonic stem cells or infected with a retroviruscontaining the transgene (except for practice of the subject inventionin avian species which is addressed elsewhere herein), the embryo isimplanted into the oviduct of a pseudopregnant female. The consequentprogeny are tested for incorporation of the transgene by Southern blotanalysis of blood or tissue samples using transgene specific probes. PCRis particularly useful in this regard. Positive progeny (G0) arecrossbred to produce offspring (G1) which are analyzed for transgeneexpression by Northern blot analysis of tissue samples.

In one aspect, the methods comprise increasing the phosphorous uptake inthe transgenic animal and/or decreasing the amount of polltant in themanure of the transgenic organism by about 15%, about 20%, or about 20%,to about 50% or more.

In one aspect, the animals contemplated for use in the practice of thesubject invention are those animals generally regarded as domesticatedanimals including pets (e.g., canines, felines, avian species etc.) andthose useful for the processing of food stuffs, i.e., avian such as meatbred and egg laying chicken and turkey, ovine such as lamb, bovine suchas beef cattle and milk cows, piscine and porcine. In one aspect, theseanimals are referred to as “transgenic” when such animal has had aheterologous DNA sequence, or one or more additional DNA sequencesnormally endogenous to the animal (collectively referred to herein as“transgenes”) chromosomally integrated into the germ cells of theanimal. The transgenic animal (including its progeny) will also have thetransgene fortuitously integrated into the chromosomes of somatic cells.

Screening Methodologies and “On-line” Monitoring Devices

In practicing the methods of the invention, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the invention, e.g., to screen polypeptides forphytaseactivity, to screen compounds as potential modulators of activity(e.g., potentiation or inhibition of enzyme activity), for antibodiesthat bind to a polypeptide of the invention, for nucleic acids thathybridize to a nucleic acid of the invention, and the like.

Immobilized Enzyme Solid Supports

The phytase enzymes, fragments thereof and nucleic acids that encode theenzymes and fragments can be affixed to a solid support. This is ofteneconomical and efficient in the use of the phytases in industrialprocesses. For example, a consortium or cocktail of phytase enzymes (oractive fragments thereof), which are used in a specific chemicalreaction, can be attached to a solid support and dunked into a processvat. The enzymatic reaction can occur. Then, the solid support can betaken out of the vat, along with the enzymes affixed thereto, forrepeated use. In one embodiment of the invention, an isolated nucleicacid of the invention is affixed to a solid support. In anotherembodiment of the invention, the solid support is selected from thegroup of a gel, a resin, a polymer, a ceramic, a glass, a microelectrodeand any combination thereof.

For example, solid supports useful in this invention include gels. Someexamples of gels include Sepharose, gelatin, glutaraldehyde,chitosan-treated glutaraldehyde, albumin-glutaraldehyde,chitosan-Xanthan, toyopearl gel (polymer gel), alginate,alginate-polylysine, carrageenan, agarose, glyoxyl agarose, magneticagarose, dextran-agarose, poly(Carbamoyl Sulfonate) hydrogel, BSA-PEGhydrogel, phosphorylated polyvinyl alcohol (PVA),monoaminoethyl-N-aminoethyl (MANA), amino, or any combination thereof.

Another solid support useful in the present invention are resins orpolymers. Some examples of resins or polymers include cellulose,acrylamide, nylon, rayon, polyester, anion-exchange resin, AMBERLITE™XAD-7, AMBERLITE™ XAD-8, AMBERLITE™ IRA-94, AMBERLITE™ IRC-50,polyvinyl, polyacrylic, polymethacrylate, or any combination thereofanother type of solid support useful in the present invention isceramic. Some examples include non-porous ceramic, porous ceramic, SiO₂,Al₂O₃. Another type of solid support useful in the present invention isglass. Some examples include non-porous glass, porous glass, aminopropylglass or any combination thereof. Another type of solid support that canbe used is a microelectrode. An example is a polyethyleneimine-coatedmagnetite. Graphitic particles can be used as a solid support. Anotherexample of a solid support is a cell, such as a red blood cell.

Methods of Immobilization

There are many methods that would be known to one of skill in the artfor immobilizing enzymes or fragments thereof, or nucleic acids, onto asolid support. Some examples of such methods include, e.g.,electrostatic droplet generation, electrochemical means, via adsorption,via covalent binding, via cross-linking, via a chemical reaction orprocess, via encapsulation, via entrapment, via calcium alginate, or viapoly (2-hydroxyethyl methacrylate). Like methods are described inMethods in Enzymology, Immobilized Enzymes and Cells, Part C. 1987.Academic Press. Edited by S. P. Colowick and N. O. Kaplan. Volume 136;and Immobilization of Enzymes and Cells. 1997. Humana Press. Edited byG. F. Bickerstaff. Series: Methods in Biotechnology, Edited by J. M.Walker.

Capillary Arrays

Capillary arrays, such as the GIGAMATRIX™, Diversa Corporation, SanDiego, Calif., can be used to in the methods of the invention. Nucleicacids or polypeptides of the invention can be immobilized to or appliedto an array, including capillary arrays. Arrays can be used to screenfor or monitor libraries of compositions (e.g., small molecules,antibodies, nucleic acids, etc.) for their ability to bind to ormodulate the activity of a nucleic acid or a polypeptide of theinvention. Capillary arrays provide another system for holding andscreening samples. For example, a sample screening apparatus can includea plurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The apparatus can further include interstitialmaterial disposed between adjacent capillaries in the array, and one ormore reference indicia formed within of the interstitial material. Acapillary for screening a sample, wherein the capillary is adapted forbeing bound in an array of capillaries, can include a first walldefining a lumen for retaining the sample, and a second wall formed of afiltering material, for filtering excitation energy provided to thelumen to excite the sample.

A polypeptide or nucleic acid, e.g., a ligand, can be introduced into afirst component into at least a portion of a capillary of a capillaryarray. Each capillary of the capillary array can comprise at least onewall defining a lumen for retaining the first component. An air bubblecan be introduced into the capillary behind the first component. Asecond component can be introduced into the capillary, wherein thesecond component is separated from the first component by the airbubble. A sample of interest can be introduced as a first liquid labeledwith a detectable particle into a capillary of a capillary array,wherein each capillary of the capillary array comprises at least onewall defining a lumen for retaining the first liquid and the detectableparticle, and wherein the at least one wall is coated with a bindingmaterial for binding the detectable particle to the at least one wall.The method can further include removing the first liquid from thecapillary tube, wherein the bound detectable particle is maintainedwithin the capillary, and introducing a second liquid into the capillarytube.

The capillary array can include a plurality of individual capillariescomprising at least one outer wall defining a lumen. The outer wall ofthe capillary can be one or more walls fused together. Similarly, thewall can define a lumen that is cylindrical, square, hexagonal or anyother geometric shape so long as the walls form a lumen for retention ofa liquid or sample. The capillaries of the capillary array can be heldtogether in close proximity to form a planar structure. The capillariescan be bound together, by being fused (e.g., where the capillaries aremade of glass), glued, bonded, or clamped side-by-side. The capillaryarray can be formed of any number of individual capillaries, forexample, a range from 100 to 4,000,000 capillaries. A capillary arraycan form a microtiter plate having about 100,000 or more individualcapillaries bound together.

Arrays, or “BioChips”

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. For example, in oneaspect of the invention, a monitored parameter is transcript expressionof a phytase gene. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array, or “biochip.” By using an “array” of nucleic acids on amicrochip, some or all of the transcripts of a cell can besimultaneously quantified. Alternatively, arrays comprising genomicnucleic acid can also be used to determine the genotype of a newlyengineered strain made by the methods of the invention. “Polypeptidearrays” can also be used to simultaneously quantify a plurality ofproteins.

In alternative aspects, “arrays” or “microarrays” or “biochips” or“chips” of the invention comprise a plurality of target elements inaddition to a nucleic acid and/or a polypeptide or peptide of theinvention; each target element can comprises a defined amount of one ormore polypeptides (including antibodies) or nucleic acids immobilizedonto a defined area of a substrate surface, as discussed in furtherdetail, below.

The present invention can be practiced with any known “array,” alsoreferred to as a “microarray” or “nucleic acid array” or “polypeptidearray” or “antibody array” or “biochip,” or variation thereof. Arraysare generically a plurality of “spots” or “target elements,” each targetelement comprising a defined amount of one or more biological molecules,e.g., oligonucleotides, immobilized onto a defined area of a substratesurface for specific binding to a sample molecule, e.g., mRNAtranscripts.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Polypeptides and Peptides

The invention provides isolated, synthetic or recombinant polypeptideshaving an amino acid sequence at least 95%, 96% 97%, 98% or 99% sequenceidentity to SEQ ID NO:2, and comprising at least one of the mutationslisted in Table 4, 5, 6, 7, 9, or any combination thereof. The inventionfurther provides isolated, synthetic or recombinant nucleic acidsencoding polypeptides having an amino acid sequence at least 95%, 96%97%, 98% or 99% sequence identity to SEQ ID NO:2, and comprising atleast one of the mutations listed in Table 4, 5, 6, 7, 9, or anycombination thereof. For reference, the synthetically generated “parent”SEQ ID NO:2 is:

Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr1               5                   10                  15Pro Gln Ser Ala Phe Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser            20                  25                  30Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr        35                  40                  45Gln Leu Met Gln Asp Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val    50                  55                  60Lys Leu Gly Glu Leu Thr Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu65                  70                  75                  80Gly His Tyr Trp Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Pro Lys                85                  90                  95Cys Gly Cys Pro Gln Ser Gly Gln Val Ala Ile Ile Ala Asp Val Asp            100                 105                 110Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro        115                 120                 125Asp Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp    130                 135                 140Pro Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala145                 150                 155                 160Asn Val Thr Asp Ala Ile Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp                165                 170                 175Phe Thr Gly His Tyr Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu            180                 185                 190Asn Phe Pro Gln Ser Asn Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu        195                 200                 205Ser Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala    210                 215                 220Asp Cys Val Ser Leu Thr Gly Ala Val Ser Leu Ala Ser Met Leu Thr225                 230                 235                 240Glu Ile Phe Leu Leu Gln Gln Ala Gln Gly Met Pro Glu Pro Gly Trp                245                 250                 255Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His            260                 265                 270Asn Ala Gln Phe Asp Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser        275                 280                 285Arg Ala Thr Pro Leu Leu Asp Leu Ile Lys Thr Ala Leu Thr Pro His    290                 295                 300Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu305                 310                 315                 320Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu                325                 330                 335Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly            340                 345                 350Gly Glu Leu Val Phe Glu Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln        355                 360                 365Trp Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp    370                 375                 380Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr385                 390                 395                 400Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala                405                 410                 415Gly Phe Thr Gln Ile Val Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu            420                 425                 430

The sequence of the parental phytase SEQ ID NO:2, encoded by, e.g., SEQID NO:1, showing the gene site saturation mutagenesis (GSSM)-generatedsequence modifications selected for GeneReassembly™ libraryconstruction, as described in Example 1, are shown in FIG. 4. Parentalphytase SEQ ID NO:2, encoded by, e.g., SEQ ID NO:1 was subjected tofurther gene site saturation mutagenesis (GSSM) sequence modifications,site directed mutagenesis (SDM) and TMCA library construction, asdescribed in Example 2.

In one aspect, polypeptide and peptides of the invention have phytaseactivity. In alternative aspects, they also can be useful as, e.g.,labeling probes, antigens, toleragens, motifs, phytase active sites.

In alternative aspects, polypeptides and peptides of the invention aresynthetic or are recombinantly generated polypeptides. Peptides andproteins can be recombinantly expressed in vitro or in vivo. Thepeptides and polypeptides of the invention can be made and isolatedusing any method known in the art. Polypeptide and peptides of theinvention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3 13) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the instructions provided by themanufacturer.

The enzymes and polynucleotides of the present invention can be providedin an isolated form or purified to homogeneity. The phytase polypeptideof the invention can be obtained using any of several standard methods.For example, phytase polypeptides can be produced in a standardrecombinant expression system (as described herein), chemicallysynthesized (although somewhat limited to small phytase peptidefragments), or purified from organisms in which they are naturallyexpressed. Useful recombinant expression methods include mammalianhosts, microbial hosts, and plant hosts.

In alternative aspects, polypeptides and peptides of the inventioncomprise “amino acids” or “amino acid sequences” that are oligopeptides,peptides, polypeptides or protein sequences, or alternatively, arefragments, portions or subunits of any of these, and to naturallyoccurring or synthetic molecules.

In alternative aspects, “recombinant” polypeptides or proteins of theinvention include (refer to) polypeptides or proteins produced byrecombinant DNA techniques; e.g., produced from cells transformed by anexogenous DNA construct encoding the desired polypeptide or protein.“Synthetic” nucleic acids (including oligonucleotides), polypeptides orproteins of the invention include those prepared by chemical synthesis,as described in detail herein. In alternative aspects, polypeptides orproteins of the invention comprise amino acids joined to each other bypeptide bonds or modified peptide bonds, i.e., peptide isosteres, andmay contain modified amino acids other than the 20 gene-encoded aminoacids. The polypeptides may be modified by either natural processes,such as post-translational processing, or by chemical modificationtechniques that are well known in the art. Modifications can occuranywhere in the polypeptide, including the peptide backbone, the aminoacid side-chains and the amino or carboxyl termini. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given polypeptide. Also agiven polypeptide may have many types of modifications, for example,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of a phosphatidylinositol,cross-linking cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristolyation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,and transfer-RNA mediated addition of amino acids to protein such asarginylation.

In alternative aspects, “synthetic” polypeptides or protein are thoseprepared by chemical synthesis. Solid-phase chemical peptide synthesismethods can also be used to synthesize the polypeptide or fragments ofthe invention. Such method have been known in the art since the early1960's (Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154, 1963) (Seealso Stewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2ed., Pierce Chemical Co., Rockford, Ill., pp. 11-12)) and have recentlybeen employed in commercially available laboratory peptide design andsynthesis kits (Cambridge Research Biochemicals). Such commerciallyavailable laboratory kits have generally utilized the teachings of H. M.Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and providefor synthesizing peptides upon the tips of a multitude of “rods” or“pins” all of which are connected to a single plate. When such a systemis utilized, a plate of rods or pins is inverted and inserted into asecond plate of corresponding wells or reservoirs, which containsolutions for attaching or anchoring an appropriate amino acid to thepin's or rod's tips. By repeating such a process step, i.e., invertingand inserting the rod and pin's tips into appropriate solutions, aminoacids are built into desired peptides. In addition, a number ofavailable FMOC peptide synthesis systems are available. For example,assembly of a polypeptide or fragment can be carried out on a solidsupport using an Applied Biosystems, Inc. Model 431A automated peptidesynthesizer. Such equipment provides ready access to the peptides of theinvention, either by direct synthesis or by synthesis of a series offragments that can be coupled using other known techniques.

In alternative aspects, peptides and polypeptides of the invention areglycosylated. The glycosylation can be added post-translationally eitherchemically or by cellular biosynthetic mechanisms, wherein the laterincorporates the use of known glycosylation motifs, which can be nativeto the sequence or can be added as a peptide or added in the nucleicacid coding sequence. The glycosylation can be O-linked or N-linked, or,a combination thereof.

In alternative aspects, peptides and polypeptides of the invention, asdefined above, comprise “mimetic” and “peptidomimetic” forms, either inpart or completely. In one aspect, the terms “mimetic” and“peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered. Thus, in one aspect, amimetic composition is within the scope of the invention if it has aphytase activity.

In alternative aspects, peptides and polypeptides of the invention havesequences comprising the specific modification to SEQ ID NO:2, asdefined above, and also conservative substitutions that may or may notmodify activity, e.g., enzymatic activity. In alternative aspects,conservative substitutions are those that substitute a given amino acidin a polypeptide by another amino acid of like characteristics. Inalternative aspects, conservative substitutions are the followingreplacements: replacements of an aliphatic amino acid such as Ala, Val,Leu and Ile with another aliphatic amino acid; replacement of a Ser witha Thr or vice versa; replacement of an acidic residue such as Asp andGlu with another acidic residue; replacement of a residue bearing anamide group, such as Asn and Gln, with another residue bearing an amidegroup; exchange of a basic residue such as Lys and Arg with anotherbasic residue; and replacement of an aromatic residue such as Phe, Tyrwith another aromatic residue.

Polypeptide mimetic compositions of the invention can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the invention include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the invention can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH2- for —C(═O)—NH—), aminomethylene (CH2-NH), ethylene, olefin(CH═CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4-), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).

A polypeptide of the invention can also be characterized as a mimetic bycontaining all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2, 3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; K- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids ornithine, citrulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,which for these reagents it may be preferable to use alkalineconditions. Tyrosine residue mimetics can be generated by reactingtyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane.N-acetylimidizol and tetranitromethane can be used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Cysteine residuemimetics can be generated by reacting cysteinyl residues with, e.g.,alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide andcorresponding amines; to give carboxymethyl or carboxyamidomethylderivatives. Cysteine residue mimetics can also be generated by reactingcysteinyl residues with, e.g., bromo-trifluoroacetone,alpha-bromo-beta-(5-imidozoyl) propionic acid; chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyldisulfide; p-chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or,chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimetics can be generated(and amino terminal residues can be altered) by reacting lysinyl with,e.g., succinic or other carboxylic acid anhydrides. Lysine and otheralpha-amino-containing residue mimetics can also be generated byreaction with imidoesters, such as methyl picolinimidate, pyridoxalphosphate, pyridoxal, chloroborohydride, trinitro-benzenesulfonic acid,O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactionswith glyoxylate. Mimetics of methionine can be generated by reactionwith, e.g., methionine sulfoxide. Mimetics of proline include, e.g.,pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxy proline,dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline.Histidine residue mimetics can be generated by reacting histidyl with,e.g., diethylprocarbonate or para-bromophenacyl bromide. Other mimeticsinclude, e.g., those generated by hydroxylation of proline and lysine;phosphorylation of the hydroxyl groups of seryl or threonyl residues;methylation of the alpha-amino groups of lysine, arginine and histidine;acetylation of the N-terminal amine; methylation of main chain amideresidues or substitution with N-methyl amino acids; or amidation ofC-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R— or S— form.

The invention also provides methods for modifying the polypeptides ofthe invention by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See, e.g.,Creighton, T. E., Proteins—Structure and Molecular Properties 2nd Ed.,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A™ automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

In one aspect, peptides and polypeptides of the invention have sequencescomprising the specific modification to SEQ ID NO:2, as defined above,and also “substantially identical” amino acid sequences, i.e., asequence that differs by one or more conservative or non-conservativeamino acid substitutions, deletions, or insertions, particularly whensuch a substitution occurs at a site that is not the active site of themolecule, and provided that the polypeptide essentially retains itsfunctional properties. In one aspect, peptides and polypeptides of theinvention have sequences comprising the specific modification to SEQ IDNO:2, as defined above, and conservative amino acid substitutions thatsubstitute one amino acid for another of the same class, for example,substitution of one hydrophobic amino acid, such as isoleucine, valine,leucine, or methionine, for another, or substitution of one polar aminoacid for another, such as substitution of arginine for lysine, glutamicacid for aspartic acid or glutamine for asparagine. In one aspect, oneor more amino acids can be deleted, for example, from a phytasepolypeptide of the invention to result in modification of the structureof the polypeptide without significantly altering its biologicalactivity, or alternative, to purposely significantly alter itsbiological activity. For example, amino- or carboxyl-terminal aminoacids that are required, or alternatively are not required, for phytasebiological activity can be removed and/or added. Modified polypeptidesequences of the invention can be assayed for phytase biologicalactivity by any number of methods, including contacting the modifiedpolypeptide sequence with a phytase substrate and determining whetherthe modified polypeptide decreases the amount of specific substrate inthe assay or increases the bioproducts of the enzymatic reaction of afunctional phytase polypeptide with the substrate.

Another aspect of the invention comprises polypeptides having about 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity to SEQ ID NO:2 and having one of thespecific enumerated sequence modifications, as discussed (set forth)above.

These amino acid sequence variants of the invention can be characterizedby a predetermined nature of the variation, a feature that sets themapart from a naturally occurring form, e.g., an allelic or interspeciesvariation of a phytase sequence. In one aspect, the variants of theinvention exhibit the same qualitative biological activity as thenaturally occurring analogue. Alternatively, the variants can beselected for having modified characteristics. In one aspect, while thesite or region for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random mutagenesis may be conducted at the target codon or regionand the expressed phytase variants screened for the optimal combinationof desired activity. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, asdiscussed herein for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants can be done using, e.g., assays ofcatalysis of phytate (myo-inositol-hexaphosphate) to inositol andinorganic phosphate; or, the hydrolysis of phytate(myo-inositol-hexaphosphate). In alternative aspects, amino acidsubstitutions can be single residues; insertions can be on the order offrom about 1 to 20 amino acids, although considerably larger insertionscan be done. Deletions can range from about 1 to about 20, 30, 40, 50,60, 70 residues or more. To obtain a final derivative with the optimalproperties, substitutions, deletions, insertions or any combinationthereof may be used. Generally, these changes are done on a few aminoacids to minimize the alteration of the molecule. However, largerchanges may be tolerated in certain circumstances.

Polypeptides of the invention may be obtained through biochemicalenrichment or purification procedures. The sequence of potentiallyhomologous polypeptides or fragments may be determined by proteolyticdigestion, gel electrophoresis and/or microsequencing.

Another aspect of the invention is an assay for identifying fragments orvariants of polypeptides of the invention. Polypeptides of the inventionmay be used to catalyze biochemical reactions to indicate that saidfragment or variant retains the enzymatic activity of a polypeptides ofthe invention.

An exemplary assay for determining if fragments of variants retain theenzymatic activity of the polypeptides of the invention comprises:contacting the polypeptide fragment or variant with a substrate moleculeunder conditions which allow the polypeptide fragment or variant tofunction, and detecting either a decrease in the level of substrate oran increase in the level of the specific reaction product of thereaction between the polypeptide and substrate.

Polypeptides of the invention may be used to catalyze biochemicalreactions. In accordance with one aspect of the invention, there isprovided a process for utilizing a polypeptide of the invention as aphytase.

The invention provides phytases having no or modified signal sequences(also called signal peptides (SPs), or leader peptides), or heterologoussignal sequences. The polypeptides of the invention also can have no ormodified or heterologous prepro domains and/or catalytic domains (CDs).The modified or heterologous SPs, prepro domains and/or CDs incorporatedin a polypeptide the invention can be part of a fusion protein, e.g., asa heterologous domain in a chimeric protein, or added by a chemicallinking agent. For example, an enzyme of the invention can comprise aheterologous SP and/or prepro in a vector, e.g., a pPIC series vector(Invitrogen, Carlsbad, Calif.).

Additionally, polypeptides of the invention can further compriseheterologous sequences, either sequences from other phytases, or fromnon-phytase sources, or entirely synthetic sequences. Thus, in oneaspect, a nucleic acid of the invention comprises coding sequence for anendogenous, modified or heterologous signal sequence (SP), prepro domainand/or catalytic domain (CD) and a heterologous sequence (i.e., asequence not naturally associated with the a signal sequence (SP),prepro domain and/or catalytic domain (CD) of the invention). Theheterologous sequence can be on the 3′ terminal end, 5′ terminal end,and/or on both ends of the SP, prepro domain and/or CD coding sequence.

Methods for identifying “prepro” domain sequences and signal sequencesare well known in the art, see, e.g., Van de Ven (1993) Crit. Rev.Oncog. 4(2):115-136. For example, to identify a prepro sequence, theprotein is purified from the extracellular space and the N-terminalprotein sequence is determined and compared to the unprocessed form.Various methods of recognition of signal sequences are known to those ofskill in the art. For example, in one aspect, signal peptides for usewith polypeptides of the invention are identified by a method referredto as SignalP. SignalP uses a combined neural network which recognizesboth signal peptides and their cleavage sites; see, e.g., Nielsen (1997)“Identification of prokaryotic and eukaryotic signal peptides andprediction of their cleavage sites” Protein Engineering 10:1-6.

The invention provides phytase enzymes where the structure of thepolypeptide backbone, the secondary or the tertiary structure, e.g., analpha-helical or beta-sheet structure, has been modified. In one aspect,the charge or hydrophobicity has been modified. In one aspect, the bulkof a side chain has been modified. Substantial changes in function orimmunological identity are made by selecting substitutions that are lessconservative. For example, substitutions can be made which moresignificantly affect: the structure of the polypeptide backbone in thearea of the alteration, for example an alpha-helical or a beta-sheetstructure; a charge or a hydrophobic site of the molecule, which can beat an active site; or a side chain. The invention provides substitutionsin polypeptide of the invention where (a) a hydrophilic residues, e.g.seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g. lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g. glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine. The variants can exhibit the same qualitative biologicalactivity (i.e., phytase enzyme activity) although variants can beselected to modify the characteristics of the phytase as needed.

In one aspect, phytases of the invention comprise epitopes orpurification tags, signal sequences or other fusion sequences, etc. Inone aspect, phytases of the invention can be fused to a random peptideto form a fusion polypeptide. By “fused” or “operably linked” herein ismeant that the random peptide and the phytase are linked together, insuch a manner as to minimize the disruption to the stability of phytaseactivity. The fusion polypeptide (or fusion polynucleotide encoding thefusion polypeptide) can comprise further components as well, includingmultiple peptides at multiple loops.

In one aspect, phytases of the invention are chimeric polypeptides,e.g., comprising heterologous SPs, carbohydrate binding modules, phytaseenzyme catalytic domains, linkers and/or non-phytase catalytic domains.The invention provides a means for generating chimeric polypeptideswhich may encode biologically active hybrid polypeptides (e.g., hybridphytase enzymes). In one aspect, the original polynucleotides encodebiologically active polypeptides. The method of the invention producesnew hybrid polypeptides by utilizing cellular processes which integratethe sequence of the original polynucleotides such that the resultinghybrid polynucleotide encodes a polypeptide demonstrating activitiesderived from the original biologically active polypeptides. For example,the original polynucleotides may encode a particular enzyme fromdifferent microorganisms. An enzyme encoded by a first polynucleotidefrom one organism or variant may, for example, function effectivelyunder a particular environmental condition, e.g. high salinity. Anenzyme encoded by a second polynucleotide from a different organism orvariant may function effectively under a different environmentalcondition, such as extremely high temperatures. A hybrid polynucleotidecontaining sequences from the first and second original polynucleotidesmay encode an enzyme which exhibits characteristics of both enzymesencoded by the original polynucleotides. Thus, the enzyme encoded by thehybrid polynucleotide may function effectively under environmentalconditions shared by each of the enzymes encoded by the first and secondpolynucleotides, e.g., high salinity and extreme temperatures.

Thus, a hybrid polypeptide resulting from this method of the inventionmay exhibit specialized enzyme activity not displayed in the originalenzymes. For example, following recombination and/or reductivereassortment of polynucleotides encoding phytase enzymes, the resultinghybrid polypeptide encoded by a hybrid polynucleotide can be screenedfor specialized enzyme activities, e.g., hydrolase, peptidase,phosphorylase, etc., activities, obtained from each of the originalenzymes. Thus, for example, the hybrid polypeptide may be screened toascertain those chemical functionalities which distinguish the hybridpolypeptide from the original parent polypeptides, such as thetemperature, pH or salt concentration at which the hybrid polypeptidefunctions.

A hybrid polypeptide resulting from the method of the invention mayexhibit specialized enzyme activity not displayed in the originalenzymes. For example, following recombination and/or reductivereassortment of polynucleotides encoding hydrolase activities, theresulting hybrid polypeptide encoded by a hybrid polynucleotide can bescreened for specialized hydrolase activities obtained from each of theoriginal enzymes, i.e., the type of bond on which the hydrolase acts andthe temperature at which the hydrolase functions. Thus, for example, aphytase may be screened to ascertain those chemical functionalitieswhich distinguish the hybrid phytase from the original phytases, suchas: (a) amide (peptide bonds), i.e., proteases; (b) ester bonds, i.e.,esterases and lipases; (c) acetals, i.e., glycosidases and, for example,the temperature, pH or salt concentration at which the hybridpolypeptide functions.

In one aspect, the invention relates to a method for producing abiologically active hybrid polypeptide and screening such a polypeptidefor enhanced activity by:

(1) introducing at least a first polynucleotide in operable linkage anda second polynucleotide in operable linkage, said at least firstpolynucleotide and second polynucleotide sharing at least one region ofpartial sequence homology, into a suitable host cell;

(2) growing the host cell under conditions which promote sequencereorganization resulting in a hybrid polynucleotide in operable linkage;

(3) expressing a hybrid polypeptide encoded by the hybridpolynucleotide;

(4) screening the hybrid polypeptide under conditions which promoteidentification of enhanced biological activity; and

(5) isolating the a polynucleotide encoding the hybrid polypeptide.

Methods for screening for various enzyme activities are known to thoseof skill in the art and are discussed throughout the presentspecification. Such methods may be employed when isolating thepolypeptides and polynucleotides of the invention.

In one aspect, the instant invention provides a method (and productsthereof) of producing stabilized aqueous liquid formulations havingphytase activity that exhibit increased resistance to heat inactivationof the enzyme activity and which retain their phytase activity duringprolonged periods of storage. The liquid formulations are stabilized bymeans of the addition of urea and/or a polyol such as sorbitol andglycerol as stabilizing agent. Also provided are feed preparations formonogastric animals and methods for the production thereof that resultfrom the use of such stabilized aqueous liquid formulations. Additionaldetails regarding this approach are in the public literature and/or areknown to the skilled artisan. In a particular non-limitingexemplification, such publicly available literature includes EP 0626010(WO 9316175 A1) (Barendse et al.), although references in the publiclyavailable literature do not teach the inventive molecules of the instantapplication.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated, synthetic or recombinant antibodiesthat specifically bind to a phytase of the invention. These antibodiescan be used to isolate, identify or quantify the phytases of theinvention or related polypeptides. These antibodies can be used toinhibit the activity of an enzyme of the invention. These antibodies canbe used to isolated polypeptides related to those of the invention,e.g., related phytase enzymes.

Antibodies of the invention can comprise a peptide or polypeptidederived from, modeled after or substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof,capable of specifically binding an antigen or epitope, see, e.g.Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press,N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush(1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

The antibodies can be used in immunoprecipitation, staining (e.g.,FACS), immunoaffinity columns, and the like. If desired, nucleic acidsequences encoding for specific antigens can be generated byimmunization followed by isolation of polypeptide or nucleic acid,amplification or cloning and immobilization of polypeptide onto an arrayof the invention. Alternatively, the methods of the invention can beused to modify the structure of an antibody produced by a cell to bemodified, e.g., an antibody's affinity can be increased or decreased.Furthermore, the ability to make or modify antibodies can be a phenotypeengineered into a cell by the methods of the invention.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

The polypeptides can be used to generate antibodies which bindspecifically to the polypeptides of the invention. The resultingantibodies may be used in immunoaffinity chromatography procedures toisolate or purify the polypeptide or to determine whether thepolypeptide is present in a biological sample. In such procedures, aprotein preparation, such as an extract, or a biological sample iscontacted with an antibody capable of specifically binding to one of thepolypeptides of the invention.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of the invention.After a wash to remove non-specifically bound proteins, the specificallybound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays, and Western Blots.

Polyclonal antibodies generated against the polypeptides of theinvention can be obtained by direct injection of the polypeptides intoan animal or by administering the polypeptides to an animal, forexample, a nonhuman. The antibody so obtained will then bind thepolypeptide itself. In this manner, even a sequence encoding only afragment of the polypeptide can be used to generate antibodies which maybind to the whole native polypeptide. Such antibodies can then be usedto isolate the polypeptide from cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique, the trioma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique (see,e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (see,e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to the polypeptides of the invention. Alternatively,transgenic mice may be used to express humanized antibodies to thesepolypeptides or fragments thereof.

Antibodies generated against the polypeptides of the invention may beused in screening for similar polypeptides from other organisms andsamples. In such techniques, polypeptides from the organism arecontacted with the antibody and those polypeptides which specificallybind the antibody are detected. Any of the procedures described abovemay be used to detect antibody binding.

Kits

The invention provides kits comprising the compositions, e.g., nucleicacids, expression cassettes, vectors, cells, polypeptides (e.g.,phytases) and/or antibodies of the invention. The kits also can containinstructional material teaching the methodologies and industrial uses ofthe invention, as described herein.

The polypeptides of the invention may also be used to generateantibodies which bind specifically to the enzyme polypeptides orfragments. The resulting antibodies may be used in immunoaffinitychromatography procedures to isolate or purify the polypeptide or todetermine whether the polypeptide is present in a biological sample. Insuch procedures, a protein preparation, such as an extract, or abiological sample is contacted with an antibody capable of specificallybinding to one of a polypeptide of SEQ ID NO:2, sequences substantiallyidentical thereto, or fragments of the foregoing sequences.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of SEQ ID NO:2,sequences substantially identical thereto, or fragment thereof. After awash to remove non-specifically bound proteins, the specifically boundpolypeptides are eluted.

The isolated polynucleotide sequences, polypeptide sequence, variantsand mutants thereof can be measured for retention of biological activitycharacteristic to the enzyme of the present invention, for example, inan assay for detecting enzymatic phytase activity (Food Chemicals Codex,4^(th) Ed.). Such enzymes include truncated forms of phytase, andvariants such as deletion and insertion variants of the polypeptidesequence as set forth in SEQ ID NO:2. These phytases havethermotolerance. That is, the phytase has a residual specific activityof about 90% after treatment at 70° C. for 30 minutes and about 50%after treatment at 75° C. for 30 minutes. The thermotolerance of theinvention phytases is advantageous in using the enzyme as a feedadditive as the feed can be molded, granulated, or pelletized at a hightemperature.

For example, in one aspect, the invention provides an edible pelletizedenzyme delivery matrix and method of use for delivery of phytase to ananimal, for example as a nutritional supplement. The enzyme deliverymatrix readily releases a phytase enzyme, such as one having the aminoacid sequence of SEQ ID NO:2 or at least 30 contiguous amino acidsthereof, in aqueous media, such as, for example, the digestive fluid ofan animal. The invention enzyme delivery matrix is prepared from agranulate edible carrier selected from such components as grain germthat is spent of oil, hay, alfalfa, timothy, soy hull, sunflower seedmeal, wheat meal, and the like, that readily disperse the recombinantenzyme contained therein into aqueous media. In use, the ediblepelletized enzyme delivery matrix is administered to an animal todelivery of phytase to the animal. Suitable grain-based substrates maycomprise or be derived from any suitable edible grain, such as wheat,corn, soy, sorghum, alfalfa, barley, and the like. An exemplarygrain-based substrate is a corn-based substrate. The substrate may bederived from any suitable part of the grain, e.g., a grain germ,approved for animal feed use, such as corn germ that is obtained in awet or dry milling process. The grain germ can comprise spent germ,which is grain germ from which oil has been expelled, such as bypressing or hexane or other solvent extraction. Alternatively, the graingerm is expeller extracted, that is, the oil has been removed bypressing.

The enzyme delivery matrix of the invention is in the form of discreteplural particles, pellets or granules. By “granules” is meant particlesthat are compressed or compacted, such as by a pelletizing, extrusion,or similar compacting to remove water from the matrix. Such compressionor compacting of the particles also promotes intraparticle cohesion ofthe particles. For example, the granules can be prepared by pelletizingthe grain-based substrate in a pellet mill. The pellets prepared therebyare ground or crumbled to a granule size suitable for use as an adjuvantin animal feed. Since the matrix is itself approved for use in animalfeed, it can be used as a diluent for delivery of enzymes in animalfeed.

The enzyme delivery matrix can be in the form of granules having agranule size ranging from about 4 to about 400 mesh (USS); or about 8 toabout 80 mesh; or about 14 to about 20 mesh. If the grain germ is spentvia solvent extraction, use of a lubricity agent such as corn oil may benecessary in the pelletizer, but such a lubricity agent ordinarily isnot necessary if the germ is expeller extracted. In other aspects of theinvention, the matrix is prepared by other compacting or compressingprocesses such as, for example, by extrusion of the grain-basedsubstrate through a die and grinding of the extrudate to a suitablegranule size.

The enzyme delivery matrix may further include a polysaccharidecomponent as a cohesiveness agent to enhance the cohesiveness of thematrix granules. The cohesiveness agent is believed to provideadditional hydroxyl groups, which enhance the bonding between grainproteins within the matrix granule. It is further believed that theadditional hydroxyl groups so function by enhancing the hydrogen bondingof proteins to starch and to other proteins. The cohesiveness agent maybe present in any amount suitable to enhance the cohesiveness of thegranules of the enzyme delivery matrix. Suitable cohesiveness agentsinclude one or more of dextrins, maltodextrins, starches, such as cornstarch, flours, cellulosics, hemicellulosics, and the like. For example,the percentage of grain germ and cohesiveness agent in the matrix (notincluding the enzyme) is 78% corn germ meal and 20% by weight of cornstarch.

Because the enzyme-releasing matrix of the invention is made frombiodegradable materials, the matrix may be subject to spoilage, such asby molding. To prevent or inhibit such molding, the matrix may include amold inhibitor, such as a propionate salt, which may be present in anyamount sufficient to inhibit the molding of the enzyme-releasing matrix,thus providing a delivery matrix in a stable formulation that does notrequire refrigeration.

The phytase enzyme contained in the invention enzyme delivery matrix andmethods is in one aspect a thermotolerant phytase, as described herein,so as to resist inactivation of the phytase during manufacture whereelevated temperatures and/or steam may be employed to prepare thepelletized enzyme delivery matrix. During digestion of feed containingthe invention enzyme delivery matrix, aqueous digestive fluids willcause release of the active enzyme. Other types of thermotolerantenzymes and nutritional supplements that are thermotolerant can also beincorporated in the delivery matrix for release under any type ofaqueous conditions.

A coating can be applied to the invention enzyme matrix particles formany different purposes, such as to add a flavor or nutrition supplementto animal feed, to delay release of animal feed supplements and enzymesin gastric conditions, and the like. Or, the coating may be applied toachieve a functional goal, for example, whenever it is desirable to slowrelease of the enzyme from the matrix particles or to control theconditions under which the enzyme will be released. The composition ofthe coating material can be such that it is selectively broken down byan agent to which it is susceptible (such as heat, acid or base, enzymesor other chemicals). Alternatively, two or more coatings susceptible todifferent such breakdown agents may be consecutively applied to thematrix particles.

The invention is also directed towards a process for preparing anenzyme-releasing matrix. In accordance with the invention, the processcomprises providing discrete plural particles of a grain-based substratein a particle size suitable for use as an enzyme-releasing matrix,wherein the particles comprise a phytase enzyme of the invention. Theprocess can include compacting or compressing the particles ofenzyme-releasing matrix into granules, which can be accomplished bypelletizing. The mold inhibitor and cohesiveness agent, when used, canbe added at any suitable time, and can be mixed with the grain-basedsubstrate in the desired proportions prior to pelletizing of thegrain-based substrate. Moisture content in the pellet mill feed can bein the ranges set forth above with respect to the moisture content inthe finished product, or about 14% to 15%, or about 10% to 20%. Moisturecan be added to the feedstock in the form of an aqueous preparation ofthe enzyme to bring the feedstock to this moisture content. Thetemperature in the pellet mill can be brought to about 82° C. withsteam. The pellet mill may be operated under any conditions that impartsufficient work to the feedstock to provide pellets. The pelletingprocess itself is a cost-effective process for removing water from theenzyme-containing composition.

In one aspect, the pellet mill is operated with a ⅛ in. by 2 in. die at100 lb./min. pressure at 82° C. to provide pellets, which then arecrumbled in a pellet mill crumbler to provide discrete plural particleshaving a particle size capable of passing through an 8 mesh screen butbeing retained on a 20 mesh screen.

The thermotolerant phytases described herein can have high optimumtemperatures and can have high heat resistance or heat tolerance. Thus,the phytases of the invention can carry out enzymatic reactions attemperatures normally considered above optimum. The phytases of theinvention also can carry out enzymatic reactions after being exposed tohigh temperatures (thermotolerance being the ability to retain enzymaticactivity at temperatures where the wild type phytase is active afterpreviously being exposed to high temperatures, even if the hightemperature can inactivate or diminish the enzyme's activity, see alsodefinition of thermotolerance, above). The gene encoding the phytaseaccording to the present invention can be used in preparation ofphytases (e.g. using GSSM and/or TMCA technology, as described herein)having characteristics different from those of the phytase of SEQ IDNO:2 (in terms of optimum pH, optimum temperature, heat resistance,stability to solvents, specific activity, affinity to substrate,secretion ability, translation rate, transcription control and thelike). Furthermore, the polynucleotides of the invention may be employedfor screening of variant phytases prepared by the methods describedherein to determine those having a desired activity, such as improved ormodified thermostability or thermotolerance. For example, U.S. Pat. No.5,830,732, describes a screening assay for determining thermotoleranceof a phytase.

An in vitro example of such a screening assay is the following assay forthe detection of phytase activity: Phytase activity can be measured byincubating 150 μl of the enzyme preparation with 600 μl of 2 mM sodiumphytate in 100 mM Tris HCl buffer, pH 7.5, supplemented with 1 mM CaCl₂for 30 minutes at 37° C. After incubation the reaction is stopped byadding 750 μl of 5% trichloroacetic acid. Phosphate released wasmeasured against phosphate standard spectrophotometrically at 700 nmafter adding 1500 μl of the color reagent (4 volumes of 1.5% ammoniummolybdate in 5.5% sulfuric acid and 1 volume of 2.7% ferrous sulfate;Shimizu, 1992). One unit of enzyme activity is defined as the amount ofenzyme required to liberate one μmol Pi per min under assay conditions.Specific activity can be expressed in units of enzyme activity per mg ofprotein. The enzyme of the present invention has enzymatic activity withrespect to the hydrolysis of phytate to inositol and free phosphate.

In one aspect, the instant invention provides a method of hydrolyzingphytate comprised of contacting the phytate with one or more of thenovel phytase molecules disclosed herein (e.g., proteins having thespecific modifications of SEQ ID NO:2). Accordingly, the inventionprovides a method for catalyzing the hydrolysis of phytate to inositoland free phosphate with release of minerals from the phytic acidcomplex. The method includes contacting a phytate substrate with adegrading effective amount of an enzyme of the invention. The term“degrading effective” amount refers to the amount of enzyme which isrequired to degrade at least 50% of the phytate, as compared to phytatenot contacted with the enzyme. 80% of the phytate can be degraded.

In another aspect, the invention provides a method for hydrolyzingphospho-mono-ester bonds in phytate. The method includes administeringan effective amount of phytase molecules of the invention, to yieldinositol and free phosphate. In one aspect, an “effective” amount refersto the amount of enzyme which is required to hydrolyze at least 50% ofthe phospho-mono-ester bonds, as compared to phytate not contacted withthe enzyme. In one aspect, at least 80% of the bonds are hydrolyzed.

In a particular aspect, when desired, the phytase molecules may be usedin combination with other reagents, such as other catalysts; in order toeffect chemical changes (e.g. hydrolysis) in the phytate moleculesand/or in other molecules of the substrate source(s). According to thisaspect, the phytase molecules and the additional reagent(s) will notinhibit each other. The phytase molecules and the additional reagent(s)can have an overall additive effect, or, alternatively, phytasemolecules and the additional reagent(s) can have an overall synergisticeffect.

Relevant sources of the substrate phytate molecules include foodstuffs,potential foodstuffs, byproducts of foodstuffs (both in vitro byproductsand in vivo byproducts, e.g. ex vivo reaction products and animalexcremental products), precursors of foodstuffs, and any other materialsource of phytate.

In a non-limiting aspect, the recombinant phytase can be consumed byorganisms and retains activity upon consumption. In anotherexemplification, transgenic approaches can be used to achieve expressionof the recombinant phytase—e.g., in a controlled fashion (methods areavailable for controlling expression of transgenic molecules intime-specific and tissue specific manners).

In one aspect, the phytase activity in the source material (e.g. atransgenic plant source or a recombinant prokaryotic host) may beincreased upon consumption; this increase in activity may occur, forexample, upon conversion of a precursor phytase molecule in pro-form toa significantly more active enzyme in a more mature form, where saidconversion may result, for example, from the ingestion and digestion ofthe phytase source. Hydrolysis of the phytate substrate may occur at anytime upon the contacting of the phytase with the phytate; for example,this may occur before ingestion or after ingestion or both before andafter ingestion of either the substrate or the enzyme or both. It isadditionally appreciated that the phytate substrate may be contactedwith—in addition to the phytase—one or more additional reagents, such asanother enzyme, which may be also be applied either directly or afterpurification from its source material.

It is appreciated that the phytase source material(s) can be contacteddirectly with the phytate source material(s); e.g. upon in vitro or invivo grinding or chewing of either or both the phytase source(s) and thephytate source(s). Alternatively the phytase enzyme may be purified awayfrom source material(s), or the phytate substrate may be purified awayfrom source material(s), or both the phytase enzyme and the phytatesubstrate may be purified away from source material(s) prior to thecontacting of the phytase enzyme with the phytate substrate. It isappreciated that a combination of purified and unpurifiedreagents—including enzyme(s) or substrates(s) or both—may be used.

It is appreciated that more than one source material may be used as asource of phytase activity. This is serviceable as one way to achieve atimed release of reagent(s) from source material(s), where release fromdifferent reagents from their source materials occur differentially, forexample as ingested source materials are digested in vivo or as sourcematerials are processed in in vitro applications. The use of more thanone source material of phytase activity is also serviceable to obtainphytase activities under a range of conditions and fluctuations thereof,that may be encountered—such as a range of pH values, temperatures,salinities, and time intervals—for example during different processingsteps of an application. The use of different source materials is alsoserviceable in order to obtain different reagents, as exemplified by oneor more forms or isomers of phytase and/or phytate and/or othermaterials.

It is appreciated that a single source material, such a transgenic plantspecies (or plant parts thereof), may be a source material of bothphytase and phytate; and that enzymes and substrates may bedifferentially compartmentalized within said single source—e.g. secretedvs. non-secreted, differentially expressed and/or having differentialabundances in different plant parts or organs or tissues or insubcellular compartments within the same plant part or organ or tissue.Purification of the phytase molecules contained therein may compriseisolating and/or further processing of one or more desirable plant partsor organs or tissues or subcellular compartments.

In one aspect, this invention provides a method of catalyzing in vivoand/or in vitro reactions using seeds containing enhanced amounts ofenzymes. The method comprises adding transgenic, non-wild type seeds,e.g., in a ground form, to a reaction mixture and allowing the enzymesin the seeds to increase the rate of reaction. By directly adding theseeds to the reaction mixture the method provides a solution to the moreexpensive and cumbersome process of extracting and purifying the enzyme.Methods of treatment are also provided whereby an organism lacking asufficient supply of an enzyme is administered the enzyme in the form ofseeds from one or more plant species, e.g., transgenic plant species,containing enhanced amounts of the enzyme. Additional details regardingthis approach are in the public literature and/or are known to theskilled artisan. In a particular non-limiting exemplification, suchpublicly available literature includes U.S. Pat. No. 5,543,576 (VanOoijen et al.) and U.S. Pat. No. 5,714,474 (Van Ooijen et al.), althoughthese reference do not teach the inventive molecules of the instantapplication and instead teach the use of fungal phytases.

In one aspect, the instant phytase molecules are serviceable forgenerating recombinant digestive system life forms (or microbes orflora) and for the administration of said recombinant digestive systemlife forms to animals. Administration may be optionally performed aloneor in combination with other enzymes and/or with other life forms thatcan provide enzymatic activity in a digestive system, where said otherenzymes and said life forms may be may recombinant or otherwise. Forexample, administration may be performed in combination with xylanolyticbacteria.

In one aspect, the present invention provides a method for steeping cornor sorghum kernels in warm water containing sulfur dioxide in thepresence of an enzyme preparation comprising one or morephytin-degrading enzymes, e.g., in such an amount that the phytinpresent in the corn or sorghum is substantially degraded. The enzymepreparation may comprise phytase and/or acid phosphatase and optionallyother plant material degrading enzymes. The steeping time may be 12 to18 hours. The steeping may be interrupted by an intermediate millingstep, reducing the steeping time. In one aspect, corn or sorghum kernelsare steeped in warm water containing sulfur dioxide in the presence ofan enzyme preparation including one or more phytin-degrading enzymes,such as phytase and acid phosphatases, to eliminate or greatly reducephytic acid and the salts of phytic acid. Additional details regardingthis approach are in the public literature and/or are known to theskilled artisan, e.g., U.S. Pat. No. 4,914,029, (Caransa et al.) and EP0321004 (Vaara et al.).

In one aspect, the present invention provides a method to obtain a breaddough having desirable physical properties such as non-tackiness andelasticity and a bread product of superior quality such as a specificvolume comprising adding phytase molecules to the bread dough. In oneaspect, phytase molecules of the instant invention are added to aworking bread dough preparation that is subsequently formed and baked.Additional details regarding this approach are in the public literatureand/or are known to the skilled artisan, for example, JP 03076529 (Haraet al.).

In one aspect, the present invention provides a method to produceimproved soybean foodstuffs. Soybeans are combined with phytasemolecules of the instant invention to remove phytic acid from thesoybeans, thus producing soybean foodstuffs that are improved in theirsupply of trace nutrients essential for consuming organisms and in itsdigestibility of proteins. In one aspect, in the production of soybeanmilk, phytase molecules of the instant invention are added to or broughtinto contact with soybeans in order to reduce the phytic acid content.In a non-limiting exemplification, the application process can beaccelerated by agitating the soybean milk together with the enzyme underheating or by a conducting a mixing-type reaction in an agitationcontainer using an immobilized enzyme. Additional details regarding thisapproach are in the public literature and/or are known to the skilledartisan, for example, JP 59166049 (Kamikubo et al.).

In one aspect, the instant invention provides a method of producing anadmixture product for drinking water or animal feed in fluid form, andwhich comprises using mineral mixtures and vitamin mixtures, and alsonovel phytase molecules of the instant invention. In a one aspect, thereis achieved a correctly dosed and composed mixture of necessarynutrients for the consuming organism without any risk of precipitationand destruction of important minerals/vitamins, while at the same timeoptimum utilization is made of the phytin-bound phosphate in the feed.Additional details regarding this approach are in the public literatureand/or are known to the skilled artisan, e.g., EP 0772978 (Bendixen etal.).

It is appreciated that the phytase molecules of the instant inventionmay also be used to produce other alcoholic and non-alcoholic drinkablefoodstuffs (or drinks) based on the use of molds and/or on grains and/oron other plants. These drinkable foodstuffs include liquors, wines,mixed alcoholic drinks (e.g. wine coolers, other alcoholic coffees suchas Irish coffees, etc.), beers, near-beers, juices, extracts,homogenates, and purees. In one aspect, the instantly disclosed phytasemolecules are used to generate transgenic versions of molds and/orgrains and/or other plants serviceable for the production of suchdrinkable foodstuffs. In another aspect, the instantly disclosed phytasemolecules are used as additional ingredients in the manufacturingprocess and/or in the final content of such drinkable foodstuffs.Additional details regarding this approach are in the public literatureand/or are known to the skilled artisan.

In one aspect, the present invention provides a means to obtain refinedsake having a reduced amount of phytin and an increased content ofinositol. Such a sake may have—through direct and/or psychogeniceffects—a preventive action on hepatic disease, arteriosclerosis, andother diseases. In one aspect, a sake is produced from rice Koji bymultiplying a rice Koji mold having high phytase activity as a rawmaterial. It is appreciated that the phytase molecules of the instantinvention may be used to produce a serviceable mold with enhancedactivity (e.g., a transgenic mold) and/or added exogenously to augmentthe effects of a Koji mold. The strain is added to boiled rice and Kojiis produced by a conventional procedure. In one exemplification, theprepared Koji is used, the whole rice is prepared at two stages and Sakeis produced at constant Sake temperature of 15° C. to give the objectiverefined Sake having a reduced amount of phytin and an increased amountof inositol. Additional details regarding this approach are in thepublic literature and/or are known to the skilled artisan, for example,JP 06153896 (Soga et al.) and JP 06070749 (Soga et al.).

In one aspect, the present invention provides a method to obtain anabsorbefacient capable of promoting the absorption of minerals includingingested calcium without being digested by gastric juices or intestinaljuices at a low cost. In one aspect, the mineral absorbefacient containsa partial hydrolysate of phytic acid as an active ingredient. A partialhydrolysate of the phytic acid can be produced by hydrolyzing the phyticacid or its salts using novel phytase molecules of the instantinvention. The treatment with the phytase molecules may occur eitheralone and/or in a combination treatment (to inhibit or to augment thefinal effect), and is followed by inhibiting the hydrolysis within arange so as not to liberate all the phosphate radicals. Additionaldetails regarding this approach are in the public literature and/or areknown to the skilled artisan, e.g., JP 04270296 (Hoshino).

In one aspect, the present invention provides a method (and productstherefrom) to produce an enzyme composition having an additive or asynergistic phytate hydrolyzing activity; said composition comprisesnovel phytase molecules of the instant invention and one or moreadditional reagents to achieve a composition that is serviceable for acombination treatment. In one aspect, the combination treatment of thepresent invention is achieved with the use of at least two phytases ofdifferent position specificity, i.e. any combinations of 1-, 2-, 3-, 4-,5-, and 6-phytases. By combining phytases of different positionspecificity an additive or synergistic effect is obtained. Compositionssuch as food and feed or food and feed additives comprising suchphytases in combination are also included in this invention as areprocesses for their preparation. Additional details regarding thisapproach are in the public literature and/or are known to the skilledartisan, e.g., WO 30681 (Ohmann et al.).

In another aspect, the combination treatment of the present invention isachieved with the use of an acid phosphatase having phytate hydrolyzingactivity at a pH of 2.5, in a low ratio corresponding to a pH 2.5:5.0activity profile of from about 0.1:1.0 to 10:1, or of from about 0.5:1.0to 5:1, or from about 0.8:1.0 to 3:1, or from about 0.8:1.0 to 2:1. Theenzyme composition can display a higher synergetic phytate hydrolyzingefficiency through thermal treatment. The enzyme composition isserviceable in the treatment of foodstuffs (drinkable and solid food,feed and fodder products) to improve phytate hydrolysis. Additionaldetails or alternative protocols regarding this approach are in thepublic literature and/or are known to the skilled artisan, e.g., U.S.Pat. No. 5,554,399 (Vanderbeke et al.) and U.S. Pat. No. 5,443,979(Vanderbeke et al.), teaching the use of fungal (in particularAspergillus) phytases.

In another aspect, the present invention provides a method (and productstherefrom) to produce a composition comprising the instant novelphytate-acting enzyme in combination with one or more additional enzymesthat act on polysaccharides. Such polysaccharides can be selected fromthe group consisting of arabinans, fructans, fucans, galactans,galacturonans, glucans, mannans, xylans, levan, fucoidan, carrageenan,galactocarolose, pectin, pectic acid, amylose, pullulan, glycogen,amylopectin, cellulose, carboxylmethylcellulose,hydroxypropylmethylcellulose, dextran, pustulan, chitin, agarose,keratan, chondroitin, dermatan, hyaluronic acid, alginic acid, andpolysaccharides containing at least one aldose, ketose, acid or amineselected from the group consisting of erythrose, threose, ribose,arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose,idose, galactose, talose, erythrulose, ribulose, xylulose, psicose,fructose, sorbose, tagatose, glucuronic acid, gluconic acid, glucaricacid, galacturonic acid, mannuronic acid, glucosamine, galactosamine andneuraminic acid.

In one aspect, the present invention provides a method (and productstherefrom) to produce a composition having a synergistic phytatehydrolyzing activity comprising one or more novel phytase molecules ofthe instant invention, a cellulase (can also include a xylanase),optionally a protease, and optionally one or more additional reagents.In alternative aspects, such combination treatments are serviceable inthe treatment of foodstuffs, wood products, such as paper products, andas cleansing solutions and solids.

In one aspect, phytases of the invention are serviceable in combinationwith cellulose components. It is known that cellulases of manycellulolytic bacteria are organized into discrete multi-enzymecomplexes, called cellulosomes. The multiple subunits of cellulosomesare composed of numerous functional domains, which interact with eachother and with the cellulosic substrate. One of these subunits comprisesa distinctive new class of non-catalytic scaffolding polypeptide, whichselectively integrates the various cellulase and xylanase subunits intothe cohesive complex. Intelligent application of cellulosome hybrids andchimeric constructs of cellulosomal domains should enable better use ofcellulosic biomass and may offer a wide range of novel applications inresearch, medicine and industry.

In one aspect, phytases of the invention are serviceable—either alone orin combination treatments—in areas of biopulping and biobleaching wherea reduction in the use of environmentally harmful chemicalstraditionally used in the pulp and paper industry is desired. Wastewater treatment represents another vast application area wherebiological enzymes have been shown to be effective not only in colorremoval but also in the bioconversion of potentially noxious substancesinto useful bioproducts.

In one aspect, phytases of the invention are serviceable for generatinglife forms that can provide at least one enzymatic activity—either aloneor in combination treatments—in the treatment of digestive systems oforganisms. Particularly relevant organisms to be treated includenon-ruminant organisms, although ruminant organisms may also benefitfrom such treatment. Specifically, it is appreciated that this approachmay be performed alone or in combination with other biological molecules(for example, xylanases) to generate a recombinant host that expresses aplurality of biological molecules. It is also appreciated that theadministration of the instant phytase molecules and/or recombinant hostsexpressing the instant phytase molecules may be performed either aloneor in combination with other biological molecules, and/or life formsthat can provide enzymatic activities in a digestive system—where saidother enzymes and said life forms may be may recombinant or otherwise.For example, administration may be performed in combination withxylanolytic bacteria.

For example, in addition to phytate, many organisms are also unable toadequately digest hemicelluloses. Hemicelluloses or xylans are majorcomponents (35%) of plant materials. For ruminant animals, about 50% ofthe dietary xylans are degraded, but only small amounts of xylans aredegraded in the lower gut of non-ruminant animals and humans. In therumen, the major xylanolytic species are Butyrivibrio fibrisolvens andBacteroides ruminicola. In the human colon, Bacteroides ovatus andBacteroides fragilis subspecies “a” are major xylanolytic bacteria.Xylans are chemically complex, and their degradation requires multipleenzymes. Expression of these enzymes by gut bacteria varies greatlyamong species. Butyrivibrio fibrisolvens makes extracellular xylanasesbut Bacteroides species have cell-bound xylanase activity. Biochemicalcharacterization of xylanolytic enzymes from gut bacteria has not beendone completely. A xylosidase gene has been cloned from B. fibrosolvens.The data from DNA hybridizations using a xylanase gene cloned from B.fibrisolvens indicate this gene may be present in other B. fibrisolvensstrains. A cloned xylanase from Bact. ruminicola was transferred to andhighly expressed in Bact. fragilis and Bact. uniformis. Arabinosidaseand xylosidase genes from Bact. ovatus have been cloned and bothactivities appear to be catalyzed by a single, bifunctional, novelenzyme.

In one aspect, phytases of the invention are serviceable for 1)transferring into a suitable host (such as Bact. fragilis or Bact.uniformis); 2) achieving adequate expression in a resultant recombinanthost; and 3) administering said recombinant host to organisms to improvethe ability of the treated organisms to degrade phytate. Continuedresearch in genetic and biochemical areas will provide knowledge andinsights for manipulation of digestion at the gut level and improvedunderstanding of colonic fiber digestion.

Additional details or alternative protocols regarding this approach arein the public literature and/or are known to the skilled artisan, forexample, the invention can incorporate procedures as described in U.S.Pat. No. 5,624,678 (Bedford et al.), U.S. Pat. No. 5,683,911 (Bodie etal.), U.S. Pat. No. 5,720,971 (Beauchemin et al.), U.S. Pat. No.5,759,840 (Sung et al.), U.S. Pat. No. 5,770,012 (Cooper), U.S. Pat. No.5,786,316 (Baeck et al.), U.S. Pat. No. 5,817,500 (Hansen et al.).

The instant invention teaches that phytase molecules of the instantinvention may be added to the reagent(s) disclosed in order to obtainpreparations having an additional phytase activity. In one aspect,reagent(s) and the additional phytase molecules will not inhibit eachother. In one aspect, the reagent(s) and the additional phytasemolecules may have an overall additive effect. In one aspect, thereagent(s) and the additional phytase molecules may have an overallsynergistic effect.

In one aspect, the present invention provides a method (and productstherefrom) for enhancement of phytate phosphorus utilization andtreatment and prevention of tibial dyschondroplasia in animals,particularly poultry, by administering to animals a feed compositioncontaining a hydroxylated vitamin D₃ derivative. The vitamin D₃derivative can be administered to animals in feed containing reducedlevels of calcium and phosphorus for enhancement of phytate phosphorusutilization. Accordingly, the vitamin D₃ derivative can be administeredin combination with novel phytase molecules of the instant invention forfurther enhancement of phytate phosphorus utilization. Additionaldetails or alternative protocols regarding this approach are in thepublic literature and/or are known to the skilled artisan, e.g., U.S.Pat. No. 5,516,525 (Edwards et al.) and U.S. Pat. No. 5,366,736 (Edwardset al.), U.S. Pat. No. 5,316,770 (Edwards et al.).

In one aspect, the present invention provides a method (and productstherefrom) to obtain foodstuff that 1) comprises phytin that is easilyabsorbed and utilized in a form of inositol in a body of an organism; 2)that is capable of reducing phosphorus in excrementary matter; and 3)that is accordingly useful for improving environmental pollution. Saidfoodstuff is comprised of an admixture of a phytin-containing grain, alactic acid-producing microorganism, and a novel phytase molecule of theinstant invention. In one aspect, said foodstuff is produced bycompounding a phytin-containing grain (e.g. rice bran) with an effectivemicrobial group having an acidophilic property, producing lactic acid,without producing butyric acid, free from pathogenicity, and a phytase.Examples of an effective microbial group include e.g. Streptomyces sp.(American Type Culture Collection No. ATCC 3004) belonging to the groupof actinomyces and Lactobacillus sp. (IFO 3070) belonging to the groupof lactobacilli.

An exemplary amount of addition of an effective microbial group is 0.2wt. % in terms of bacterial body weight based on a grain material. Inone aspect, the amount of the addition of the phytase is about 1-2 wt. %based on the phytin in the grain material. Additional details oralternative protocols regarding this approach are in the publicliterature and/or are known to the skilled artisan, e.g., JP 08205785(Akahori et al.).

In one aspect, the present invention provides a method for improving thesolubility of vegetable proteins. More specifically, the inventionrelates to methods for the solubilization of proteins in vegetableprotein sources, which methods comprise treating the vegetable proteinsource with an efficient amount of one or more phytase enzymes of theinvention and treating the vegetable protein source with an efficientamount of one or more proteolytic enzymes. In another aspect, theinvention provides animal feed additives comprising a phytase of theinvention and one or more proteolytic enzymes. Additional details oralternative protocols regarding this approach are in the publicliterature and/or are known to the skilled artisan, e.g., EP 0756457 (WO9528850 A1) (Nielsen and Knap).

In one aspect, the present invention provides a method of producing aplant protein preparation comprising dispersing vegetable protein sourcematerials in water at a pH in the range of 2 to 6 and admixing phytasemolecules of the instant invention therein. The acidic extractcontaining soluble protein is separated and dried to yield a solidprotein of desirable character. One or more proteases can also be usedto improve the characteristics of the protein. Additional details oralternative protocols regarding this approach are in the publicliterature and/or are known to the skilled artisan, e.g., U.S. Pat. No.3,966,971.

In one aspect, the present invention provides a method (and productsthereof) to activate inert phosphorus in soil and/or compost, to improvethe utilization rate of a nitrogen compound, and to suppress propagationof pathogenic molds by adding three reagents, phytase, saponin andchitosan, to the compost.

In one aspect, the method can comprise treating the compost by 1) addingphytase-containing microorganisms in media, e.g., recombinant hosts thatoverexpress the novel phytase molecules of the instant invention, forexample, at 100 ml media/100 kg wet compost; 2) alternatively alsoadding a phytase-containing plant source—such as wheat bran—e.g. at 0.2to 1 kg/100 kg wet compost; 3) adding a saponin-containing source—suchas peat, mugworts and yucca plants—e.g. at 0.5 to 3.0 g/kg; 4) addingchitosan-containing materials—such as pulverized shells of shrimps,crabs, etc.—e.g. at 100 to 300 g/kg wet compost.

In one aspect, recombinant sources the three reagents, phytase, saponin,and chitosan, are used. Additional details or alternative protocolsregarding this approach are in the public literature and/or are known tothe skilled artisan, e.g., JP 07277865 (Toya Taisuke).

In some instances it may be advantageous to deliver and express aphytase sequence of the invention locally (e.g., within a particulartissue or cell type). For example, local expression of a phytase ordigestive enzyme in the gut of an animal will assist in the digestionand uptake of, for example, phytate and phosphorous, respectively. Thenucleic sequence may be directly delivered to the salivary glands,tissue and cells and/or to the epithelial cells lining the gut, forexample. Such delivery methods are known in the art and includeelectroporation, viral vectors and direct DNA uptake. Any polypeptidehaving phytase activity can be utilized in the methods of the invention(e.g., those specifically described under this subsection 6.3.18, aswell as those described in other sections of the invention).

For example, a nucleic acid constructs of the present invention willcomprise nucleic acid molecules in a form suitable for uptake intotarget cells within a host tissue. The nucleic acids may be in the formof bare DNA or RNA molecules, where the molecules may comprise one ormore structural genes, one or more regulatory genes, antisense strands,strands capable of triplex formation, or the like. Commonly, the nucleicacid construct will include at least one structural gene under thetranscriptional and translational control of a suitable regulatoryregion. More usually, nucleic acid constructs of the present inventionwill comprise nucleic acids incorporated in a delivery vehicle toimprove transfection efficiency, wherein the delivery vehicle will bedispersed within larger particles comprising a dried hydrophilicexcipient material.

One such delivery vehicles comprises viral vectors, such asretroviruses, adenoviruses, and adeno-associated viruses, which havebeen inactivated to prevent self-replication but which maintain thenative viral ability to bind a target host cell, deliver geneticmaterial into the cytoplasm of the target host cell, and promoteexpression of structural or other genes which have been incorporated inthe particle. Suitable retrovirus vectors for mediated gene transfer aredescribed in Kahn et al. (1992) Circ. Res. 71:1508-1517. A suitableadenovirus gene delivery is described in Rosenfeld et al. (1991) Science252:431-434. Both retroviral and adenovirus delivery systems aredescribed in Friedman (1989) Science 244:1275-1281.

A second type of nucleic acid delivery vehicle comprises liposomaltransfection vesicles, including both anionic and cationic liposomalconstructs. The use of anionic liposomes requires that the nucleic acidsbe entrapped within the liposome. Cationic liposomes do not requirenucleic acid entrapment and instead may be formed by simple mixing ofthe nucleic acids and liposomes. The cationic liposomes avidly bind tothe negatively charged nucleic acid molecules, including both DNA andRNA, to yield complexes which give reasonable transfection efficiency inmany cell types. See, Farhood et al. (1992) Biochem. Biophys. Acta.1111:239-246. An exemplary material for forming liposomal vesicles islipofectin which is composed of an equimolar mixture ofdioleylphosphatidyl ethanolamine (DOPE) anddioleyloxypropyl-triethylammonium (DOTMA), as described in Feigner andRingold (1989) Nature 337:387-388.

It is also possible to combine these two types of delivery systems. Forexample, Kahn et al. (1992), supra., teaches that a retrovirus vectormay be combined in a cationic DEAE-dextran vesicle to further enhancetransformation efficiency. It is also possible to incorporate nuclearproteins into viral and/or liposomal delivery vesicles to even furtherimprove transfection efficiencies. See, Kaneda et al. (1989) Science243:375-378.

In another aspect, a digestive aid containing an enzyme either as thesole active ingredient or in combination with one or more other agentsand/or enzymes is provided. The use of enzymes and other agents indigestive aids of livestock or domesticated animals not only improvesthe animal's health and life expectancy but also assists in increasingthe health of livestock and in the production of foodstuffs fromlivestock.

The invention also can use feeds for livestock (e.g., certain poultryfeed) that are highly supplemented with numerous minerals (e.g.,inorganic phosphorous), enzymes, growth factors, drugs, and other agentsfor delivery to the livestock. These supplements replace many of thecalories and natural nutrients present in grain, for example. Byreducing or eliminating the inorganic phosphorous supplement and othersupplements (e.g., trace mineral salts, growth factors, enzymes,antibiotics) from the feed itself, the feed is able to carry morenutrient and energy. Accordingly, the remaining diet would contain moreusable energy. For example, grain-oilseed meal diets generally containabout 3,200 kcal metabolizable energy per kilogram of diet, and mineralsalts supply no metabolizable energy. Removal of the unneeded mineralsand substitution with grain therefore increase the usable energy in thediet. Thus, the invention is differentiated over commonly used phytasecontaining feed. For example, in one aspect, a biocompatible material isused that is resistant to digestion by the gastrointestinal tract of anorganism.

In many organisms, including, for example, poultry or birds such as, forexample, chickens, turkeys, geese, ducks, parrots, peacocks, ostriches,pheasants, quail, pigeons, emu, kiwi, loons, cockatiel, cockatoo,canaries, penguins, flamingoes, and dove, the digestive tract includes agizzard which stores and uses hard biocompatible objects (e.g., rocksand shells from shell fish) to help in the digestion of seeds or otherfeed consumed by a bird. A typical digestive tract of this generalfamily of organisms, includes the esophagus which contains a pouch,called a crop, where food is stored for a brief period of time. From thecrop, food moves down into the true stomach, or proventriculus, wherehydrochloric acid and pepsin starts the process of digestion. Next, foodmoves into the gizzard, which is oval shaped and thick walled withpowerful muscles. The chief function of the gizzard is to grind or crushfood particles—a process which is aided by the bird swallowing smallamounts of fine gravel or grit. From the gizzard, food moves into theduodenum. The small intestine of birds is similar to mammals. There aretwo blind pouches or ceca, about 4-6 inches in length at the junction ofthe small and large intestine. The large intestine is short, consistingmostly of the rectum about 3-4 inches in length. The rectum empties intothe cloaca and feces are excreted through the vent.

Hard, biocompatible objects consumed (or otherwise introduced) andpresented in the gizzard provide a useful vector for delivery of variousenzymatic, chemical, therapeutic and antibiotic agents. These hardsubstances have a life span of a few hours to a few days and are passedafter a period of time. Accordingly, the invention provides coated,impregnated (e.g., impregnated matrix and membranes) modified dietaryaids for delivery of useful digestive or therapeutic agents to anorganism. Such dietary aids include objects which are typically ingestedby an organism to assist in digestion within the gizzard (e.g., rocks orgrit). The invention provides biocompatible objects that have coatedthereon or impregnated therein agents useful as a digestive aid for anorganism or for the delivery of a therapeutic or medicinal agent orchemical.

In one aspect, the invention provides a dietary aid, having abiocompatible composition designed for release of an agent that assistsin digestion, wherein the biocompatible composition is designed for oralconsumption and release in the digestive tract (e.g., the gizzard) of anorganism. “Biocompatible” means that the substance, upon contact with ahost organism (e.g., a bird), does not elicit a detrimental responsesufficient to result in the rejection of the substance or to render thesubstance inoperable. Such inoperability may occur, for example, byformation of a fibrotic structure around the substance limitingdiffusion of impregnated agents to the host organism therein or asubstance which results in an increase in mortality or morbidity in theorganism due to toxicity or infection. A biocompatible substance may benon-biodegradable or biodegradable. In one aspect, the biocompatiblecomposition is resistant to degradation or digestion by thegastrointestinal tract. In another aspect, the biocompatible compositionhas the consistency of a rock or stone.

A non-biodegradable material useful in the invention is one that allowsattachment or impregnation of a dietary agent. Such non-limitingnon-biodegradable materials include, for example, thermoplastics, suchas acrylic, modacrylic, polyamide, polycarbonate, polyester,polyethylene, polypropylene, polystyrene, polysulfone, polyethersulfone,and polyvinylidene fluoride. Elastomers are also useful materials andinclude, for example, polyamide, polyester, polyethylene, polypropylene,polystyrene, polyurethane, polyvinyl alcohol and silicone (e.g.,silicone based or containing silica). The invention provides that thebiocompatible composition can contain a plurality of such materials,which can be, e.g., admixed or layered to form blends, copolymers orcombinations thereof.

In one aspect, a “biodegradable” material means that the compositionwill erode or degrade in vivo to form smaller chemical species.Degradation may occur, for example, by enzymatic, chemical or physicalprocesses. Suitable biodegradable materials contemplated for use in theinvention include, but are not limited to, poly(lactide)s,poly(glycolide)s, poly(lactic acid)s, poly(glycolic acid)s,polyanhydrides, polyorthoesters, polyetheresters, polycaprolactone,polyesteramides, polycarbonate, polycyanoacrylate, polyurethanes,polyacrylate, and the like. Such materials can be admixed or layered toform blends, copolymers or combinations thereof.

In one aspect, a number different biocompatible substances of theinvention may be given to the animal and ingested sequentially, orotherwise provided to the same organism simultaneously, or in variouscombinations (e.g., one material before the other). In addition, thebiocompatible substances of the invention may be designed for slowpassage through the digestive tract. For example, large or fattysubstances tend to move more slowly through the digestive tract,accordingly, a biocompatible material having a large size to preventrapid passing in the digestive tract can be used. Such large substancescan be a combination of non-biodegradable and biodegradable substances.For example, a small non-biodegradable substance can be encompassed by abiodegradable substance of the invention such that over a period of timethe biodegradable portion will be degraded allowing thenon-biodegradable portion to pass through the digestive trace. Inaddition, it is recognized that any number of flavorings can be providedto a biocompatible substance of the invention to assist in consumption.

Any number of agents alone or in combination with other agents can becoated on the biocompatible substances of the invention, includingpolypeptides (e.g., enzymes, antibodies, cytokines or therapeutic smallmolecules), and antibiotics, for example. Examples of particular usefulagents are listed in Table 1 and 2, below. It is also contemplated thatcells can be encapsulated into the biocompatible material of theinvention and used to deliver the enzymes or therapeutics. For example,porous substances can be designed that have pores large enough for cellsto grow in and through and that these porous materials can then be takeninto the digestive tract. For example, the biocompatible substance ofthe invention can comprise a plurality of microfloral environments(e.g., different porosity, pH etc.) that provide support for a pluralityof cell types. The cells can be genetically engineered to deliver aparticular drug, enzyme or chemical to the organism. The cells can beeukaryotic or prokaryotic.

TABLE 1 Treatment Class Chemical Description Antibiotics Amoxycillin andIts Combination Treatment Against Bacterial Diseases Caused MastoxInjection By Gram + and Gram − Bacteria (Amoxycillin and Cloxacillin)Ampicillin and Its Combination Treatment Against Bacterial DiseasesCaused Biolox Injection By Gram + And Gram − Bacteria. (Ampicillin andCloxacillin) Nitrofurazone + Urea Treatment Of Genital Infections NefreaBolus Trimethoprim + Treatment Of Respiratory Tract Infections,Sulphamethoxazole Gastro Intestinal Tract Infections, Urino-GenitalTrizol Bolus Infections. Metronidazole and Furazolidone Treatment OfBacterial And Protozoal Diseases. Metofur Bolus Phthalylsulphathiazole,Pectin and Treatment Of Bacterial And Non-Specific Kaolin Diarrhoea,Bacillary Dysentery And Calf Pectolin Scours. Bolus SuspensionAntihelmintics Ectoparasiticide Ectoparasiticide and Antiseptic GermexOintment (Gamma Benzene Hexachloride, Proflavin Hemisulphate andCetrimide) Endoparasiticides > Albendazole Prevention And Treatment OfRoundworm, and Its Combination Tapeworm and Fluke Infestations Alben(Albendazole) Suspension (Albendazole 2.5%) Plus Suspension (Albendazole5%) Forte Bolus (Albendazole 1.5 Gm.) Tablet (Albendazole 600 Mg.)Powder (Albendazole 5%, 15%) Alpraz (Albendazole and Prevention AndTreatment Of Roundworm and Praziquantel)Tablet Tapeworm Infestation InCanines and Felines. Oxyclozanide and Its Combination Prevention andTreatment Of Fluke Infestations Clozan (Oxyclozanide) Bolus, SuspensionTetzan (Oxyclozanide and Prevention and Treatment Of Roundworm andTetramisole Hcl) Bolus, Fluke Infestations Suspension Fluzan(Oxyclozanide and Prevention and Treatment Of Roundworm Levamisole Hcl)Bolus, Infestations and Increasing Immunity Suspension LevamisolePrevention and Treatment Of Roundworm Nemasol Injection Infestations andIncreasing Immunity. Wormnil Powder Fenbendazole Prevention AndTreatment of Roundworm and Fenzole Tapeworm Infestations Tablet(Fenbendazole150 Mg.) Bolus (Fenbendazole 1.5 Gm.) Powder (Fenbendazole2.5% W/W) Tonics Vitamin B Complex, Amino Acids Treatment Of Anorexia,Hepatitis, Debility, and Liver Extract Neuralgic Convulsions Emaciationand Stunted Heptogen Injection Growth. Calcium Levulinate With Vit.B₁₂Prevention and treatment of hypocalcaemia, and Vit D₃ supportive therapyin sick conditions (especially Hylactin Injection hypothermia) andtreatment of early stages of rickets. Animal Feed Essential Minerals,Selenium and Treatment Of Anoestrus Causing Infertility and SupplementsVitamin E Repeat Breeding In Dairy Animals and Horses. Gynolactin BolusEssential Minerals, Vitamin E, and Infertility, Improper Lactation,Decreased Iodine Immunity, Stunted Growth and Debility. Hylactin PowderEssential Electrolytes With Diarrhoea, Dehydration, Prior to and afterVitamin C Transportation, In Extreme temperatures (High Electra - CPowder Or Low) and other Conditions of stress. Pyrenox Plus (DiclofenacSodium + Treatment Of Mastitis, Pyrexia Post Surgical Paracetamol)Bolus, Injection. Pain and Inflammation, Prolapse Of Uterus, Lamenessand Arthritis.

TABLE 2 Therapeutic Formulations Product Description Acutrim ®Once-daily appetite suppressant tablets. (phenylpropanolamine) TheBaxter ® Infusor For controlled intravenous delivery of anticoagulants,antibiotics, chemotherapeutic agents, and other widely used drugs.Catapres-TTS ® (clonidine Once-weekly transdermal system for thetreatment of hypertension. transdermal therapeutic system) Covera HS3(verapamil Once-daily Controlled-Onset Extended-Release (COER-24)tablets for hydrochloride) the treatment of hypertension and anginapectoris. DynaCirc CR ® (isradipine) Once-daily extended release tabletsfor the treatment of hypertension. Efidac 24 ® (chlorpheniramineOnce-daily extended release tablets for the relief of allergy symptoms.maleate) Estraderm ® Twice-weekly transdermal system for treatingcertain postmenopausal (estradiol transdermal system) symptoms andpreventing osteoporosis Glucotrol XL ® (glipizide) Once-daily extendedrelease tablets used as an adjunct to diet for the control ofhyperglycemia in patients with non-insulin-dependent diabetes mellitus.IVOMEC SR ® Bolus Ruminal delivery system for season-long control ofmajor internal and (ivermectin) external parasites in cattle. MinipressXL ® (prazosin) Once-daily extended release tablets for the treatment ofhypertension. NicoDerm ® CQ ™ (nicotine Transdermal system used as aonce-daily aid to smoking cessation for transdermal system) relief ofnicotine withdrawal symptoms. Procardia XL ® (nifedipine) Once-dailyextended release tablets for the treatment of angina and hypertension.Sudafed ® 24 Hour Once-daily nasal decongestant for relief of colds,sinusitis, hay fever (pseudoephedrine) and other respiratory allergies.Transderm-Nitro ® Once-daily transdermal system for the prevention ofangina pectoris (nitroglycerin transdermal due to coronary arterydisease. system) Transderm Scop ® (scopolamin Transdermal system for theprevention of nausea and vomiting transdermal system) associated withmotion sickness. Volmax (albuterol) Extended release tablets for reliefof bronchospasm in patients with reversible obstructive airway disease.Actisite ® (tetracycline hydrochloride) Periodontal fiber used as anadjunct to scaling and root planing for reduction of pocket depth andbleeding on probing in patients with adult periodontitis. ALZET ®Osmotic pumps for laboratory research. Amphotec ® (amphotericin BAMPHOTEC ® is a fungicidal treatment for invasive aspergillosis incholesteryl sulfate complex for patients where renal impairment orunacceptable toxicity precludes use injection) of amphotericin B ineffective doses and in patients with invasive aspergillosis where prioramphotericin B therapy has failed. BiCitra ® (sodium citrate andAlkalinizing agent used in those conditions where long-term citric acid)maintenance of alkaline urine is desirable. Ditropan ® (oxybutynin Forthe relief of symptoms of bladder instability associated with chloride)uninhibited neurogenic or reflex neurogenic bladder (i.e., urgency,frequency, urinary leakage, urge incontinence, dysuria). Ditropan ® XL(oxybutynin is a once-daily controlled-release tablet indicated for thetreatment of chloride) overactive bladder with symptoms of urge urinaryincontinence, urgency and frequency. DOXIL ® (doxorubicin HCl liposomeinjection) Duragesic ® (fentanyl 72-hour transdermal system formanagement of chronic pain in transdermal system) CII patients whorequire continuous opioid analgesia for pain that cannot be managed bylesser means such as acetaminophen-opioid combinations, non-steroidalanalgesics, or PRN dosing with short- acting opioids. Elmiron ®(pentosan polysulfate Indicated for the relief of bladder pain ordiscomfort associated with sodium) interstitial cystitis. ENACTAirWatch ™ An asthma monitoring and management system. Ethyol ®(amifostine) Indicated to reduce the cumulative renal toxicityassociated with repeated administration of cisplatin in patients withadvanced ovarian cancer or non-small cell lung cancer. Indicated toreduce the incidence of moderate to severe xerostomia in patientsundergoing post-operative radiation treatment for head and neck cancer,where the radiation port includes a substantial portion of the parotidglands. Mycelex ® Troche (clotrimazole) For the local treatment oforopharyngeal candidiasis. Also indicated prophylactically to reduce theincidence of oropharyngeal candidiasis in patients immunocompromised byconditions that include chemotherapy, radiotherapy, or steroid therapyutilized in the treatment of leukemia, solid tumors, or renaltransplantation. Neutra-Phos ® (potassium and a dietary/nutritionalsupplement sodium phosphate) PolyCitra ®-K Oral Solution Alkalinizingagent useful in those conditions where long-term and PolyCitra ®-KCrystals maintenance of an alkaline urine is desirable, such as inpatents with (potassium citrate and citric uric acid and cystine calculiof the urinary tract, especially when the acid) administration of sodiumsalts is undesirable or contraindicated PolyCitra ®-K Syrup and LCAlkalinizing agent useful in those conditions where long-term(tricitrates) maintenance of an alkaline urine is desirable, such as inpatients with uric acid and cystine calculi of the urinary tract.Progestasert ® (progesterone) Intrauterine Progesterone ContraceptiveSystem Testoderm ® Testoderm ® with Testosterone Transdermal SystemAdhesive and Testoderm ® TTS The Testoderm ® products are indicated forreplacement therapy in CIII males for conditions associated with adeficiency or absence of endogenous testosterone: (1) Primaryhypogonadism (congenital or acquired) or (2) Hypogonadotropichypogonadism (congenital or acquired). Viadur ™ (leuprolide acetateOnce-yearly implant for the palliative treatment of prostate cancerimplant)

Certain agents can be designed to become active or in activated undercertain conditions (e.g., at certain pH's, in the presence of anactivating agent etc.). In addition, it may be advantageous to usepro-enzymes in the compositions of the invention. For example, apro-enzymes can be activated by a protease (e.g., a salivary proteasethat is present in the digestive tract or is artificially introducedinto the digestive tract of an organism). It is contemplated that theagents delivered by the biocompatible compositions of the invention areactivated or inactivated by the addition of an activating agent whichmay be ingested by, or otherwise delivered to, the organism. Anothermechanism for control of the agent in the digestive tract is anenvironment sensitive agent that is activated in the proper digestivecompartment. For example, an agent may be inactive at low pH but activeat neutral pH. Accordingly, the agent would be inactive in the gut butactive in the intestinal tract. Alternatively, the agent can becomeactive in response to the presence of a microorganism specific factor(e.g., microorganisms present in the intestine).

In one aspect, the potential benefits of the present invention include,for example, (1) reduction in or possible elimination of the need formineral supplements (e.g., inorganic phosphorous supplements), enzymes,or therapeutic drugs for animal (including fish) from the daily feed orgrain thereby increasing the amount of calories and nutrients present inthe feed, and (2) increased health and growth of domestic andnon-domestic animals including, for example, poultry, porcine, bovine,equine, canine, and feline animals.

A large number of enzymes can be used in the methods and compositions ofthe present invention in addition to the phytases of the invention.These enzymes include enzymes necessary for proper digestion of consumedfoods, or for proper metabolism, activation or derivation of chemicals,prodrugs or other agents or compounds delivered to the animal via thedigestive tract. Examples of enzymes that can be delivered orincorporated into the compositions of the invention, include, forexample, feed enhancing enzymes selected from the group consisting ofα-galactosidases, β-galactosidases, in particular lactases, phytases,β-glucanases, in particular endo-β-1,4-glucanases andendo-β-1,3(4)-glucanases, cellulases, xylosidases, galactanases, inparticular arabinogalactan endo-1,4-β-galactosidases and arabinogalactanendo-1,3-β-galactosidases, endoglucanases, in particularendo-1,2-β-glucanase, endo-1,3-α-glucanase, and endo-1,3-β-glucanase,pectin degrading enzymes, in particular pectinases, pectinesterases,pectin lyases, polygalacturonases, arabinanases, rhamnogalacturonases,rhamnogalacturonan acetyl esterases, rhamnogalacturonan-α-rhamnosidase,pectate lyases, and α-galacturonisidases, mannanases, β-mannosidases,mannan acetyl esterases, xylan acetyl esterases, proteases, xylanases,arabinoxylanases and lipolytic enzymes such as lipases, phytases andcutinases. Phytases in addition to the phytases having an amino acidsequence as set forth in SEQ ID NO:2 can be used in the methods andcompositions of the invention.

In one aspect, the enzyme used in the compositions (e.g., a dietary aid)of the present invention is a phytase enzyme which is stable to heat andis heat resistant and catalyzes the enzymatic hydrolysis of phytate,i.e., the enzyme is able to renature and regain activity after a brief(i.e., 5 to 30 seconds), or longer period, for example, minutes orhours, exposure to temperatures of above 50 C.

A “feed” and a “food,” respectively, means any natural or artificialdiet, meal or the like or components of such meals intended or suitablefor being eaten, taken in, digested, by an animal and a human being,respectively. “Dietary Aid,” as used herein, denotes, for example, acomposition containing agents that provide a therapeutic or digestiveagent to an animal or organism. A “dietary aid,” typically is not asource of caloric intake for an organism, in other words, a dietary aidtypically is not a source of energy for the organism, but rather is acomposition which is taken in addition to typical “feed” or “food”.

In various aspects of the invention, feed composition are provided thatcomprise a recombinant phytase protein having at least thirty contiguousamino acids of a protein having an amino acid sequence of SEQ ID NO:2;and a phytate-containing foodstuff. As will be known to those skilled inthe art, such compositions may be prepared in a number of ways,including but not limited to, in pellet form with or without polymercoated additives, in granulate form, and by spray drying. By way ofnon-limiting example, teachings in the art directed to the preparationof feed include International Publication Nos. WO0070034 A1, WO0100042A1, WO0104279 A1, WO0125411 A1, WO0125412 A1, and EP 1073342A.

An agent or enzyme (e.g., a phytase) may exert its effect in vitro or invivo, i.e. before intake or in the stomach or gizzard of the organism,respectively. Also a combined action is possible.

Although any enzyme may be incorporated into a dietary aid, reference ismade herein to phytase as an exemplification of the methods andcompositions of the invention. A dietary aid of the invention includesan enzyme (e.g., a phytase). Generally, a dietary aid containing aphytase composition is liquid or dry.

Liquid compositions need not contain anything more than the enzyme (e.g.a phytase), preferably in a highly purified form. Usually, however, astabilizer such as glycerol, sorbitol or mono propylene glycol is alsoadded. The liquid composition may also comprise other additives, such assalts, sugars, preservatives, pH-adjusting agents, proteins, phytate (aphytase substrate). Typical liquid compositions are aqueous or oil-basedslurries. The liquid compositions can be added to a biocompatiblecomposition for slow release. Preferably the enzyme is added to adietary aid composition that is a biocompatible material (e.g.,biodegradable or non-biodegradable) and includes the addition ofrecombinant cells into, for example, porous microbeads.

Dry compositions may be spray dried compositions, in which case thecomposition need not contain anything more than the enzyme in a dryform. Usually, however, dry compositions are so-called granulates whichmay readily be mixed with a food or feed components, or more preferably,form a component of a pre-mix. The particle size of the enzymegranulates preferably is compatible with that of the other components ofthe mixture. This provides a safe and convenient means of incorporatingenzymes into animal feed. Granulates of the invention can bebiocompatible, or they can be biocompatible granulates that arenon-biodegradable.

Agglomeration granulates of the invention coated by an enzyme can beprepared using agglomeration technique in a high shear mixer. Absorptiongranulates are prepared by having cores of a carrier material toabsorb/be coated by the enzyme. In one aspect, the carrier material is abiocompatible non-biodegradable material that simulates the role ofstones or grit in the gizzard of an animal. Typical filler materialsused in agglomeration techniques include salts, such as disodiumsulphate. Other fillers are kaolin, talc, magnesium aluminum silicateand cellulose fibers. Optionally, binders such as dextrins are alsoincluded in agglomeration granulates. The carrier materials can be anybiocompatible material including biodegradable and non-biodegradablematerials (e.g., rocks, stones, ceramics, various polymers). In oneaspect, the granulates are coated with a coating mixture. Such mixturecomprises coating agents, e.g., hydrophobic coating agents, such ashydrogenated palm oil and beef tallow, and if desired other additives,such as calcium carbonate or kaolin.

In one aspect, the dietary aid compositions (e.g., phytase dietary aidcompositions) may contain other substituents such as coloring agents,aroma compounds, stabilizers, vitamins, minerals, other feed or foodenhancing enzymes etc. In one aspect, an additive used in a compositionof the invention comprises one or more compounds such as vitamins,minerals or feed enhancing enzymes and suitable carriers and/orexcipients.

In one aspect, the dietary aid compositions of the inventionadditionally comprise an effective amount of one or more feed enhancingenzymes, in particular feed enhancing enzymes selected from the groupconsisting of α-galactosidases, β-galactosidases, in particularlactases, other phytases, β-glucanases, in particularendo-β-1,4-glucanases and endo-β-1,3(4)-glucanases, cellulases,xylosidases, galactanases, in particular arabinogalactanendo-1,4-β-galactosidases and arabinogalactan endo-1,3-β-galactosidases,endoglucanases, in particular endo-1,2-β-glucanase,endo-1,3-α-glucanase, and endo-1,3-β-glucanase, pectin degradingenzymes, in particular pectinases, pectinesterases, pectin lyases,polygalacturonases, arabinanases, rhamnogalacturonases,rhamnogalacturonan acetyl esterases, rhamnogalacturonan-α-rhamnosidase,pectate lyases, and α-galacturonisidases, mannanases, β-mannosidases,mannan acetyl esterases, xylan acetyl esterases, proteases, xylanases,arabinoxylanases and lipolytic enzymes such as lipases, phytases andcutinases.

The animal dietary aid of the invention is supplemented to themono-gastric animal before or simultaneously with the diet. In oneaspect, the dietary aid of the invention is supplemented to themono-gastric animal simultaneously with the diet. In another aspect, thedietary aid is added to the diet in the form of a granulate or astabilized liquid.

An effective amount of an enzyme in a dietary aid of the invention isfrom about 10-20,000; from about 10 to 15,000, from about 10 to 10,000,from about 100 to 5,000, or from about 100 to about 2,000 FYT/kg dietaryaid.

Non-limiting examples of other specific uses of the phytase of theinvention is in soy processing and in the manufacture of inositol orderivatives thereof.

The invention also relates to a method for reducing phytate levels inanimal manure, wherein the animal is fed a dietary aid containing aneffective amount of the phytase of the invention. As stated in thebeginning of the present application one important effect thereof is toreduce the phosphate pollution of the environment.

In another aspect, the dietary aid is a magnetic carrier. For example, amagnetic carrier containing an enzyme (e.g., a phytase) distributed in,on or through a magnetic carrier (e.g., a porous magnetic bead), can bedistributed over an area high in phytate and collected by magnets aftera period of time. Such distribution and recollection of beads reducesadditional pollution and allows for reuse of the beads. In addition, useof such magnetic beads in vivo allows for the localization of thedietary aid to a point in the digestive tract where, for example,phytase activity can be carried out. For example, a dietary aid of theinvention containing digestive enzymes (e.g., a phytase) can belocalized to the gizzard of the animal by juxtapositioning a magnet nextto the gizzard of the animal after the animal consumes a dietary aid ofmagnetic carriers. The magnet can be removed after a period of timeallowing the dietary aid to pass through the digestive tract. Inaddition, the magnetic carriers are suitable for removal from theorganism after sacrificing or to aid in collection.

When the dietary aid is a porous particle, such particles are typicallyimpregnated by a substance with which it is desired to release slowly toform a slow release particle. Such slow release particles may beprepared not only by impregnating the porous particles with thesubstance it is desired to release, but also by first dissolving thedesired substance in the first dispersion phase. In this case, slowrelease particles prepared by the method in which the substance to bereleased is first dissolved in the first dispersion phase are alsowithin the scope and spirit of the invention. The porous hollowparticles may, for example, be impregnated by a slow release substancesuch as a medicine, agricultural chemical or enzyme. In particular, whenporous hollow particles impregnated by an enzyme are made of abiodegradable polymers, the particles themselves may be used as anagricultural chemical or fertilizer, and they have no adverse effect onthe environment. In one aspect the porous particles are magnetic innature.

The porous hollow particles may be used as a bioreactor support, inparticular an enzyme support. Therefore, it is advantageous to preparethe dietary aid utilizing a method of a slow release, for instance byencapsulating the enzyme of agent in a microvesicle, such as a liposome,from which the dose is released over the course of several days,preferably between about 3 to 20 days. Alternatively, the agent (e.g.,an enzyme) can be formulated for slow release, such as incorporationinto a slow release polymer from which the dosage of agent (e.g.,enzyme) is slowly released over the course of several days, for examplefrom 2 to 30 days and can range up to the life of the animal.

In one aspect, liposomes of the invention are derived from phospholipidsor other lipid substances. Liposomes are formed by mono- ormultilamellar hydrated liquid crystals that are dispersed in an aqueousmedium. Any non-toxic, physiologically acceptable and metabolizablelipid capable of forming liposomes can be used. The compositions of theinvention in liposome form can contain stabilizers, preservatives,excipients, and the like in addition to the agent. Some exemplary lipidsare the phospholipids and the phosphatidyl cholines (lecithins), bothnatural and synthetic. Methods to form liposomes are known in the art.See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV,Academic Press, New York, N.Y. (1976), p. 33 et seq.

Also within the scope of the invention is the use of a phytase of theinvention during the preparation of food or feed preparations oradditives, i.e., the phytase exerts its phytase activity during themanufacture only and is not active in the final food or feed product.This aspect is relevant for instance in dough making and baking.Accordingly, phytase or recombinant yeast expressing phytase can beimpregnated in, on or through a magnetic carriers, distributed in thedough or food medium, and retrieved by magnets.

The dietary aid of the invention may be administered alone to animals ina biocompatible (e.g., a biodegradable or non-biodegradable) carrier orin combination with other digestion additive agents. The dietary aid ofthe invention thereof can be readily administered as a top dressing orby mixing them directly into animal feed or provided separate from thefeed, by separate oral dosage, by injection or by transdermal means orin combination with other growth related edible compounds, theproportions of each of the compounds in the combination being dependentupon the particular organism or problem being addressed and the degreeof response desired. It should be understood that the specific dietarydosage administered in any given case will be adjusted in accordancewith the specific compounds being administered, the problem to betreated, the condition of the subject and the other relevant facts thatmay modify the activity of the effective ingredient or the response ofthe subject, as is well known by those skilled in the art. In general,either a single daily dose or divided daily dosages may be employed, asis well known in the art.

If administered separately from the animal feed, forms of the dietaryaid can be prepared by combining them with non-toxic pharmaceuticallyacceptable edible carriers to make either immediate release or slowrelease formulations, as is well known in the art. Such edible carriersmay be either solid or liquid such as, for example, corn starch,lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil andpropylene glycol. If a solid carrier is used the dosage form of thecompounds may be tablets, capsules, powders, troches or lozenges or topdressing as micro-dispersible forms. If a liquid carrier is used, softgelatin capsules, or syrup or liquid suspensions, emulsions or solutionsmay be the dosage form. The dosage forms may also contain adjuvants,such as preserving, stabilizing, wetting or emulsifying agents, solutionpromoters, etc. They may also contain other therapeutically valuablesubstances. A process for preparing a granulate edible carrier at hightemperature for release of enzyme when ingested is described incopending U.S. patent application Ser. No. 09/910,579, filed Jul. 20,2001.

In alternative embodiments, significant advantages of the invention mayinclude 1) ease of manufacture of the active ingredient loadedbiocompatible compositions; 2) versatility as it relates to the class ofpolymers and/or active ingredients which may be utilized; 3) higheryields and loading efficiencies; and 4) the provision of sustainedrelease formulations that release active, intact active agents in vivo,thus providing for controlled release of an active agent over anextended period of time. In one embodiment, an advantage may be due tothe local delivery of the agent with in the digestive tract (e.g., thegizzard) of the organism. In one aspect, the phrase “contained within”denotes a method for formulating an agent into a composition useful forcontrolled release, over an extended period of time of the agent.

In alternative embodiments of the sustained-release or slow releasecompositions of the invention an effective amount of an agent (e.g., anenzyme or antibiotic) is utilized. In one aspect, sustained release orslow release refers to the gradual release of an agent from abiocompatible material, over an extended period of time. The sustainedrelease can be continuous or discontinuous, linear or non-linear, andthis can be accomplished using one or more biodegradable ornon-biodegradable compositions, drug loadings, selection of excipients,or other modifications. However, it is to be recognized that it may bedesirable to provide for a “fast” release composition that provides forrapid release once consumed by the organism. It is also to be understoodthat “release” does not necessarily mean that the agent is released fromthe biocompatible carrier. Rather in one aspect, the slow releaseencompasses slow activation or continual activation of an agent presenton the biocompatible composition. For example, a phytase need not bereleased from the biocompatible composition to be effective. In thisaspect, the phytase is immobilized on the biocompatible composition.

The animal feed may be any protein-containing organic meal normallyemployed to meet the dietary requirements of animals. Many of suchprotein-containing meals are typically primarily composed of corn,soybean meal or a corn/soybean meal mix. For example, typicalcommercially available products fed to fowl include Egg Maker Complete,a poultry feed product of Land O'Lakes AG Services, as well as CountryGame and Turkey Grower a product of Agwa, Inc. (see also The EmuFarmer's Handbook by Phillip Minnaar and Maria Minnaar). Both of thesecommercially available products are typical examples of animal feedswith which the present dietary aid and/or the enzyme phytase may beincorporated to reduce or eliminate the amount of supplementalphosphorus, zinc, manganese and iron intake required in suchcompositions.

The invention provides novel formulations and dietary supplements andadditives, and methods for diet supplementation for certain diets, e.g.,Atkins' diet, vegetarian diet, macrobiotic diet, vegan diet or regionaldiets, e.g., developing world diets. Foods associated with certainelective diets, such as Atkins, vegetarian, macrobiotic, vegan orregional diets (for example, developing world diets) emphasize certainfood categories, such as proteins and fats, soy, etc., or they rely onindigenous crops, e.g., cereals, rice, beans, and the like assubstantial or sole contributors to individual nutrition. Many of thesecereal based crops have elevated (3 to 10 fold) levels of phytic acid.Processed food products such as soy protein hydrolysate and othersappear to retain elevated levels of phytic acid and their inclusion as aprotein source to nutrient bars, powders and other foods or foodsupplements and ingredients increases the phytic acid load experiencedby individuals who practice these diets.

Preventing and Reversing Bone Loss

The invention also provides novel pharmaceutical and dietaryformulations to be used as supplements and additives, and methods fordietary supplementation, comprising phytases, e.g., any phytase,including a phytase of the invention, for individuals predisposed tobone loss, individuals with bone loss, and individuals with certainmedical conditions, e.g., osteoporosis, cachexia, and medicaltreatments, such as chemotherapies, which can compromise the properuptake or utilization of essential nutrients. The methods andcompositions of the invention can be used alone or in combination withother supplements or treatment regimens, including with medications andthe like. For example, the formulations, dietary supplements and methodsfor diet supplementation can be administered with other dietarysupplements or medications for the treatment or prevention ofosteoporosis, e.g., with vitamin D3 and/or calcium (which are proven inpreventing bone loss). In one aspect, the invention provides aformulation comprising a phytase, e.g., any phytase or a phytase of theinvention, and vitamin D3 and/or calcium. In one aspect, the inventionprovides a formulation comprising a phytase, e.g., any phytase or aphytase of the invention, for preventing bone loss. In one aspect, theinvention provides a formulation comprising a phytase, e.g., any phytaseor a phytase of the invention, for reversing bone loss.

The formulation can be in the form of a pharmaceutical composition, or,can be an additive to a pharmaceutical, either of which can be inliquid, solid, powder, lotion, spray or aerosol forms. Pharmaceuticalcompositions and formulations of the invention for oral administrationcan be formulated using pharmaceutically acceptable carriers well knownin the art in appropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

The invention provides aqueous suspensions comprising a phytase, e.g., aphytase of the invention, in admixture with excipients suitable for themanufacture of aqueous suspensions. Such excipients include a suspendingagent, such as sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agents' rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617;Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995)Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108;the latest edition of Remington, The Science and Practice of Pharmacy20^(th) Ed. Lippincott Williams & Wilkins). The state of the art allowsthe clinician to determine the dosage regimen for each individualpatient, active agent and disease or condition treated. Guidelinesprovided for similar compositions used as pharmaceuticals can be used asguidance to determine the dosage regiment, i.e., dose schedule anddosage levels, administered practicing the methods of the invention(e.g., reversing bone loss, or, preventing bone loss) are appropriateand correct.

Physical Training Supplements

The invention also provides novel dietary supplements and additives, andmethods of using them, comprising phytases, e.g., any phytase, or, aphytase of the invention, for individuals undergoing athletic or otherintense physical training, e.g., training for soldiers. Athletictraining and hyperexertion can deplete essential nutrients and requiredietary supplementation. These diets and conditions have in common alack of essential micronutrients such as metals (K, Ca, Fe, Zn, Mn, Se)and ions (PO₄) necessary for optimal nutrition. Diets rich in phyticacid exacerbate this problem and may also lead to both chronic and acuteconditions that result from either voluntary or economically enforceddependence on diets rich in high phytic acid foods.

For example, individuals following various low carbohydrate (“low carb”)diets are often plagued with muscle, e.g., leg muscle, cramps. Typicaladvice for this is to add additional potassium, calcium and othernutrients to their diet. This invention provides compositions fordietary supplementation, dietary aids and supplements and methods fordiet supplementation to enhance otherwise compromised nutrition via themobilization of macro and micronutrients using phytase supplementationto the diet (including use of any phytase, or, a phytase of theinvention).

In one aspect of the invention, the use of a phytase (e.g., use of anyphytase, or, a phytase of the invention) is optimized to demonstratethermo labile or pH-stability profiles that will make it suitable foraddition directly to the food and supplement process and/or demonstrateenhanced stability and activity in the human or animal gastro intestinaltract.

The invention also provides novel dietary supplements and additives, andmethods of using them, comprising phytases, e.g., any phytase, or, aphytase of the invention, for individuals undergoing mineralsupplementation. Mineral supplementation for people on foods with highphytic acid content may actually exacerbate problems with nutrientavailability. Literature references suggest that complexes of phyticacid, calcium and zinc are much more insoluble that complexes of phyticacid and calcium. People often take multi mineral supplements. Theaddition of phytase to a scheme devised to combine mineral supplementsin the presence of high phytic acid foods could make these supplementsmuch more effective.

In alternative aspects, the compositions and methods of the invention(comprising any phytase, or, a phytase of the invention) are used assupplements or additives to

-   -   Weight-loss programs which limit intake of particular food        groups, vegetarian, macrobiotic or vegan diets which limit or        preclude intake of meats, nightshade vegetables, breads, etc and        other diets which focus on intake of nuts,    -   Specific supplement for individuals on low carb diets rich in        high phytic acid foods to ease physiological symptoms based on        reduced mineral uptake,    -   Athletic training regimens which seek to enhance performance        through dietary intake, including military training regimens,    -   Hospital diets tailored to specific needs of patients        compromised in uptake or restricted to food groups    -   Micronutrient-poor cereal and legume diets in the developing        world,    -   School lunch programs.

The invention also provides kits comprising compositions of theinvention (comprising any phytase, or, a phytase of the invention) andinstructions on incorporating the composition or method of the inventioninto these diets. The kits can comprise any packaging, labeling, productinserts and the like.

In one aspect, the invention provides a natural phytase or an optimizedphytase of the invention, formulated for or optimized for (e.g.,sequence optimized for) production, processing or passage thru human oranimal system, e.g., digestive tract. The phytase enzyme can beoptimized using alternative formulations.

Alternatively, a phytase enzyme of the invention, or, any phytase, canbe optimized by engineering of its sequence, e.g., using for example,directed evolution, error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, ligation reassembly,GSSM™ and any combination thereof, to retain activity during processing,ingestion and in the human gut.

The compositions (e.g., dietary formulations comprising any phytaseenzyme, or a phytase enzyme of the invention) can be delivered in anumber of ways to provide dietary efficacy. For example, the inventionprovides compositions (e.g., dietary formulations or additivescomprising any phytase enzyme, or a phytase enzyme of the invention) andmethods comprising use of:

-   -   In packaged food supplements such as chewable tablets or        nutritional bars,    -   As a lyophilized product available for hydration prior to        ingestion,    -   Co-packaged with dietary products, eg., processed soy product or        sold as a formulation with soybean protein hydrolysate and other        processing fractions from whole foods that are sold as        ingredients to the processed food industry,    -   In commercial baked goods,    -   Spray-on to breakfast cereals,    -   Spray-administered (e.g., nasal spray) formulations,    -   As a transgenic product expressed in indigenous crops, ie.,        cereals and legumes (e.g., as a transgenic product of a        microorganism, such as a bacterium)    -   As a transgenic organism, e.g., a microorganism; for example, a        human or animal is fed a bacteria or other microorganism capable        of making (and, in an alternative embodiment, secreting) a        recombinant phytase, such as a phytase of the invention, after        ingestion or implantation, e.g., into the gut of the human or        animal.

Phytase-containing products and methods of the invention can be brandedas nutrient enhanced, nutrient compatible or otherwise noted for anability to enhance nutrient performance and relieve various symptomsassociated with nutrient deficiency.

Phytase-containing products and methods of the invention are used tomitigate the anti-nutritive effects of phytate, which chelates importantdietary minerals such as zinc, copper, iron, magnesium, tin, andcalcium. According, phytase-containing products and methods of theinvention are used as dietary supplements to prevent the precipitationof metal-binding enzymes and proteins in ingested foods. In one aspect,the phytase-containing products and methods of the invention are used tomitigate the anti-nutritive effects of phytate in human diets, inparticular those rich in legumes and cereals, to increase mineralbioavailability. In one aspect, a phytase in a dietary supplement of theinvention catalyzes the partial or complete hydrolytic removal oforthophosphate from a phytate, where complete hydrolysis of phytateresults in the production of 1 molecule of inositol and 6 molecules ofinorganic phosphate.

Phytase-containing products and methods of the invention are applicableto the diet of humans and numerous animals, including fowl and fish. Forexample, phytase-containing dietary supplement products and dietarysupplement methods of the invention can be practiced with commerciallysignificant species, e.g., pigs, cattle, sheep, goats, laboratoryrodents (rats, mice, hamsters and gerbils), fur-bearing animals such asmink and fox, and zoo animals such as monkeys and apes, as well asdomestic mammals such as cats and dogs. Typical commercially significantavian species include chickens, turkeys, ducks, geese, pheasants, emu,ostrich, loons, kiwi, doves, parrots, cockatiel, cockatoo, canaries,penguins, flamingoes, and quail. Commercially farmed fish such as troutwould also benefit from the dietary aids disclosed herein. Other fishthat can benefit include, for example, fish (especially in an aquariumor aquaculture environment, e.g., tropical fish), goldfish and otherornamental carp, catfish, trout, salmon, shark, ray, flounder, sole,tilapia, medaka, guppy, molly, platyfish, swordtail, zebrafish, andloach.

Phytase-containing products and methods of the invention are also usedin various agars, gels, medias, and solutions used in tissue and/or cellculturing. Inconsistent soy hydrolysates can be a problem encounteredwhen using tissue and/or cell culturing. In one aspect,phytase-containing products and methods of the invention are used ascell culture media additives or as treatments to, e.g., increase cellculture yield and performance consistency. In one aspect, the inventionprovides hydrolysate for cell culturing comprising phytases, e.g.,phytases of the invention.

In one aspect, to provide a consistent product, the invention providemethods for making hydrolysates, supplements or other additives for cellculturing comprising phytases by using phytase biomarkers. For example,the method would comprise “scoring” or “marking” several molecules ofphytase in batches of hydrolysate, supplement or other additive, andthen blending the batches in the hydrolysate, supplement or otheradditive to achieve a consistent biomarker pattern. In one aspect,culture performance with each batch is measured in a mini-bioreactor(s)and performance with each biomarker and batch is correlated. In oneaspect, a blend is made to generate a higher performance product that isconsistent or better than average. In one aspect, thioredoxin (TRX) isadded to increase the bioavailability of many proteins by eliminatingsecondary structure caused by disulfide bonds. In one aspect, proteasesare also added to the hydrolysates, supplements or additives of theinvention. The proteases can be “scored” or quality controlled withother biomarkers (as with phytase, as discussed above) to direct theblending process.

In one aspect, the invention provides methods for adding phytases tograins to provide a consistent product using a biomarker “scoring” orquality control process analogous to that described above for thehydrolysates, supplements or additives of the invention.

Enzyme Enhanced Diets for Increased Warfighter Efficiency and Morale

In one aspect, the invention provides novel dietary supplements andadditives and methods for diet supplementation comprising phytases,e.g., any phytase, or, a phytase of the invention, for enzyme-enhanceddiets for increased warfighter efficiency and morale. In one aspect,these dietary supplement compositions of the invention work, in situ, toenhance energy, stamina and morale in a stable, easily usable anddesirable format while limiting food waste.

In one aspect, these dietary supplement compositions and methods of theinvention address the military operational challenge comprisingefficient delivery of nutrients and the associated health, morale andoperational effectiveness of soldiers. The invention provides enzymesoptimized to function efficiently in the human gut. These enzymes canenhance extraction of nutrients and generation of energy as well asprolong maintenance of nutritional sufficiency and individual satiety.

In addition to phytase, other enzymes, e.g., amylases, xylanases,proteases, lipases, are used to practice the dietary supplementcompositions and methods of the invention. In one aspect, the inventionprovides formulations, food supplements, foods, self-contained mealReady-to-Eat units (MREs), drinks, hydrating agents and the like,comprising phytase, e.g., a phytase of the invention, and anotherenzyme, e.g., amylases, xylanases, proteases, lipases or a combinationthereof. When ingested with food, these enzymes have been shown toenhance the release of critical nutrients, e.g., phosphorus, essentialmetals and ions, amino acids, and sugars. Furthermore, co-ingestion ofthese enzymes increases gastrointestinal mechanics and absorption bydepolymerizing plant-derived cellulose, hemicellulose and starch. Thiswhite paper proposes the development of these enzymes as supplements tomilitary diets to provide enhanced nutrient utilization for warfighter.

In one aspect, the food supplement of the invention causes the releaseof essential phosphate from normally anti-nutritive, plant-derivedphytate to increase food energy yield and bone CaPO₄ deposition. In oneaspect, phytases and other potential nutritional supplement enzymes canwithstand gut pH and endogenous protease activities.

In one aspect, the invention provides enzyme supplements to rations,drinks, foods, MREs, hydrating agents and the like to significantlyimprove nutritional value, digestibility and energy content of militarymeals (or any meal, including general consumer meal and dietsupplementation products) served to warfighters in training, battle orany stressful situation. The supplement can be formulated for ease ofuse and personal transport (in or with MREs, hydrating agents, etc). Inone aspect, the enzyme supplement will not compromise food appearance,taste and/or consistency. In one aspect, the product improve health andincreases the stamina of warfighters.

In alternative aspects, the enzyme supplement is delivered in a numberof ways to provide dietary efficacy; for example, the invention providesphytases, including phytases of the invention, and in some aspects,additional enzymes, in:

-   -   Packaged food or drink supplements such as MREs, rations,        survival kits, hydrating agents, chewable tablets or nutritional        bars;    -   As a lyophilized product (e.g., a powder) available for        hydration prior to ingestion;    -   Co-packaged with dietary products, foods, drinks, e.g.,        processed soy product or a formulation with soybean protein        hydrolysate and other processing fractions from whole foods that        are sold as ingredients to the processed food industry;    -   In baked goods;    -   Spray-on to cereals;    -   Formulations such as tablets, geltabs, capsules, sprays and the        like.

In one aspect, the compositions and methods of the invention providenutritional supplementation that rapidly releases calories and macro-and micronutrients from ingested meals. In one aspect, the compositionsand methods of the invention provides energy and body strength toindividuals in stressful situations, e.g., involving hyperexertion anddiscontinuous periods of depravation. In one aspect, the compositionsand methods of the invention provide enzymes optimized and formulated towork effectively in human gut while maintaining stability, shelf lifeand transportability in a desired environment, e.g., a military setting.

In one aspect, the compositions and methods of the invention provideformulations for increased taste characteristics, dissolvability,chewability and personal transport efficiency of the product. In oneaspect, the compositions and methods of the invention further compriseother components, such as potassium, glucose, CaCl₂. CaCl₂ in theformulation can combine with released phosphate and, in turn, enhancebone deposition and weight gain. In one aspect, the compositions andmethods of the invention further comprise formulations of other enzymes,such as proteases, cellulases, hemicellulases, for protein, celluloseand hemicellulose digestion, respectively. These enzymes can improveprotein and starch availability and further increase iron absorptionfrom many iron-rich foods.

In one aspect, the compositions and methods of the invention furthercomprise enzymes for hydrolyzing foods derived from plant material,which is rich in the glucose and xylose-based polymers, cellulose,hemicellulose and starch, as well as in the amino acid polymer, protein.In one aspect, the compositions and methods of the invention facilitatehydrolysis of polymeric materials in foods; i.e., to facilitate completedigestion polymers to monomers, e.g., polysaccharides to monomericsugars, or proteins to amino acid moieties. Thus, in this aspect, thecompositions and methods of the invention allow a food, drink or rationto realize its full caloric and nutritional value. In one aspect, enzymesupplementation comprises use of stabile enzymes, e.g., hydrolases ofvarious kinds, cellulases, hemicellulases, amylases, lipases, amidases,proteases and other enzymes. In one aspect, enzymes used in thecompositions and methods of the invention can withstand ambient gutconditions, i.e., stability at low pH and in the presence of gastricproteases.

Industrial Uses of Phytases

In addition to those described above, the invention provides novelindustrial uses for phytases, including use of the novel phytases of theinvention.

Reducing Phosphate Pollution in the Environment

In one aspect, the invention provides compositions comprising phytases(including the phytases of the invention) for addition to waste ormanure piles to convert “environmental” phytic acid. In one aspect, thisserves the purposes of reducing pollution and increasing nutrientavailability. The invention also provides compositions and methods foradding a phytase to soil, natural or artificial bodies of water (e.g.,lakes, ponds, wells, manure ponds, and the like), municipal sewage, anysewage effluent, and the like. As described above, the inventionprovides compositions and methods for reducing phytate levels in wasteor sewage, e.g., an animal manure, wherein the animal is fed a dietaryaid containing an effective amount of a phytase, e.g., a phytase of theinvention. An exemplary application of the compositions and methods ofthe invention is to reduce the phosphate pollution in the environment.Thus, the compositions and methods of the invention can be used in anyapplication that reduces pollution by degrading phytic acids.

Farming and Plant Growth Applications

In one aspect, the invention provides compositions comprising phytases(including the phytases of the invention) and methods for farmingapplications or other plant growth applications, e.g., adding phytasesto fertilizers or plant food additives (e.g., MIRACLEGROW™) for plants,e.g., house plants. In using the compositions and methods of theinvention for farming applications, users include organic farmers. Thecompositions and methods of the invention can be used for addingphytases to any soil deficient in phosphorous or needing supplementaryphosphorous for a particular crop or application. Because phosphorousrelease helps plants grow, compositions and methods of the invention canbe used for adding phytases to anything that has algae or plant materialin it.

Products of Manufacture

The invention provides a variety of products of manufacture comprisingone or more phytases of this invention. For example, in one aspect, theinvention provides compositions comprising phytases (including thephytases of the invention) and methods for cosmetic applications, e.g.,shampoos, lotions or soaps containing plant products.

In one aspect, the invention provides compositions comprising phytases(including the phytases of the invention) and methods for immobilizingthe phytase. In one aspect, the immobilized phytase acts as a controlledrelease mechanism. For example, in one aspect, the invention providescontrol released (time release) formulations of phytases for applicationto soil, e.g., clay, to house plants, etc. In one aspect, the phytasesare immobilized to beads, e.g., polysorb beads. These beads can bedelivered to soil, e.g., for agricultural or house plants. In anotheraspect, control released (time release) formulations of phytases of theinvention are used in dietary supplements and additives.

Biofuels and Biomass Conversion

The invention provides methods for making fuels, e.g., biofuels,comprising use of one or more phytases of this invention; includingproviding fuels, e.g., biofuels, comprising one or more phytases of thisinvention. The invention provides methods for biomass conversioncomprising use of one or more phytases of this invention.

In one aspect, the invention provides compositions comprising phytases(including the phytases of the invention) and methods for using thephytase in a fermentation or alcohol production process, e.g. ethanolproduction. For example, the compositions and methods of the inventioncan be used to provide effective and sustainable alternatives oradjuncts to use of petroleum-based products, e.g., as a mixture ofbioethanol and gasoline.

The invention provides organisms expressing enzymes of the invention forparticipation in chemical cycles involving natural biomass conversion.In addition, the combination of phytase (e.g., an enzyme of thisinvention) with one or more starch degrading enzymes, such as amylase orglucoamylase, improves the production of ethanol from starch. Theinvention provides methods for discovering and implementing the mosteffective of enzymes to enable these important new “biomass conversion”and alternative energy industrial processes.

Biomass Conversion and Production of Clean Bio Fuels

The invention provides polypeptides, including enzymes (phytases of theinvention) and antibodies, and methods for the processing of a biomassor any lignocellulosic material (e.g., any composition comprising acellulose, hemicellulose and lignin), to a fuel (e.g., a bioethanol,biopropanol, biobutanol, biopropanol, biomethanol, biodiesel), inaddition to feeds, foods and chemicals. For example, in one aspect, anenzyme of the invention breaks down undigestable phytic acid (phytate)in a biomass (e.g., a lignocellulosic material, a grain or an oil seed)to release digestible phosphorus; thus, in one embodiment, phytases ofthis invention are used to treat or pretreat a biomass.

Thus, the compositions and methods of the invention can be used in theproduction and/or processing of biofuels, e.g., to provide effective andsustainable alternatives and/or adjuncts to use of petroleum-basedproducts; for example, compositions and methods of the invention can beused with a mixture of enzymes to produce a biofuel—such as biomethanol,bioethanol, biopropanol, biobutanol, biodiesel and the like; which canbe added to a diesel fuel, a gasoline, a kerosene and the like. Theinvention provides organisms expressing enzymes of the invention forparticipation in chemical cycles involving natural biomass conversion.In one aspect, enzymes and methods for the conversion are used in enzymeensembles for the efficient processing of biomass in conjunction withthe depolymerization of polysaccharides, cellulosic and/orhemicellulosic polymers to metabolizeable (e.g., fermentable) carbonmoieties. The invention provides methods for discovering andimplementing the most effective of enzymes to enable these important new“biomass conversion” and alternative energy industrial processes.

The compositions and methods of the invention can be used to provideeffective and sustainable alternatives or adjuncts to use ofpetroleum-based products, e.g., as a mixture of bioethanol, biopropanol,biobutanol, biopropanol, biomethanol and/or biodiesel and gasoline. Theinvention provides organisms expressing enzymes of the invention forparticipation in chemical cycles involving natural biomass conversion.The invention provides methods for discovering and implementing the mosteffective of enzymes to enable these important new “biomass conversion”and alternative energy industrial processes.

The invention provides methods, enzymes and mixtures of enzymes or“cocktails” of the invention, for processing a material, e.g. a biomassmaterial, e.g., compositions comprising a cellooligsaccharide, anarabinoxylan oligomer, a lignin, a lignocellulose, a xylan, a glucan, acellulose and/or a fermentable sugar; e.g., including methods comprisingcontacting the composition with a polypeptide of the invention, or apolypeptide encoded by a nucleic acid of the invention, whereinoptionally the material is derived from an agricultural crop (e.g.,wheat, barley, potatoes, switchgrass, poplar wood), is a byproduct of afood or a feed production, is a lignocellulosic waste product, or is aplant residue or a waste paper or waste paper product, and optionallythe plant residue comprise stems, leaves, hulls, husks, corn or corncobs, corn stover, corn fiber, hay, straw (e.g. rice straw or wheatstraw), sugarcane bagasse, sugar beet pulp, citrus pulp, and citruspeels, wood, wood thinnings, wood chips, wood pulp, pulp waste, woodwaste, wood shavings and sawdust, construction and/or demolition wastesand debris (e.g. wood, wood shavings and sawdust), and optionally thepaper waste comprises discarded or used photocopy paper, computerprinter paper, notebook paper, notepad paper, typewriter paper,newspapers, magazines, cardboard and paper-based packaging materials,and recycled paper materials. In addition, urban wastes, e.g. the paperfraction of municipal solid waste, municipal wood waste, and municipalgreen waste, along with other materials containing sugar, starch, and/orcellulose can be used. In alternative embodiments, the processing of thematerial, e.g. the biomass material, generates a bioalcohol, e.g., abiodiesel, bioethanol, biomethanol, biobutanol or biopropanol.

Alternatively, the polypeptide of the invention may be expressed in thebiomass plant material or feedstock itself.

The methods of the invention also include taking the convertedlignocellulosic material (processed by enzymes of the invention) andmaking it into a fuel (e.g. a bioalcohol, e.g., a bioethanol,biomethanol, biobutanol or biopropanol, or biodiesel) by fermentationand/or by chemical synthesis. In one aspect, the produced sugars arefermented and/or the non-fermentable products are gasified.

The methods of the invention also include converting algae, virginvegetable oils, waste vegetable oils, animal fats and greases (e.g.tallow, lard, and yellow grease), or sewage, using enzymes of theinvention, and making it into a fuel (e.g. a bioalcohol, e.g., abioethanol, biomethanol, biobutanol or biopropanol, or biodiesel) byfermentation and/or by chemical synthesis or conversion.

The enzymes of the invention (including, for example, organisms, such asmicroorganisms, e.g., fungi, yeast or bacteria, making and in someaspects secreting recombinant enzymes of the invention) can be used inor included/integrated at any stage of any biomass conversion process,e.g., at any one step, several steps, or included in all of the steps,or all of the following methods of biomass conversion processes, or allof these biofuel alternatives:

-   -   Direct combustion: the burning of material by direct heat and is        the simplest biomass technology; can be very economical if a        biomass source is nearby.    -   Pyrolysis: is the thermal degradation of biomass by heat in the        absence of oxygen. In one aspect, biomass is heated to a        temperature between about 800 and 1400 degrees Fahrenheit, but        no oxygen is introduced to support combustion resulting in the        creation of gas, fuel oil and charcoal.    -   Gasification: biomass can be used to produce methane through        heating or anaerobic digestion. Syngas, a mixture of carbon        monoxide and hydrogen, can be derived from biomass.    -   Landfill Gas: is generated by the decay (anaerobic digestion) of        buried garbage in landfills. When the organic waste decomposes,        it generates gas consisting of approximately 50% methane, the        major component of natural gas.    -   Anaerobic digestion: converts organic matter to a mixture of        methane, the major component of natural gas, and carbon dioxide.        In one aspect, biomass such as waterwaste (sewage), manure, or        food processing waste, is mixed with water and fed into a        digester tank without air.    -   Fermentation        -   Alcohol Fermentation: fuel alcohol is produced by converting            cellulosic mass and/or starch to sugar, fermenting the sugar            to alcohol, then separating the alcohol water mixture by            distillation. Feedstocks such as dedicated crops (e.g.,            corn, wheat, barley, potatoes, switchgrass, Miscanthus,            poplar wood), agricultural residues and wastes (e.g. rice            straw, corn stover, wheat straw, sugarcane bagasse, rice            hulls, corn fiber, sugar beet pulp, citrus pulp, and citrus            peels), forestry wastes (e.g. hardwood and softwood            thinnings, hardwood and softwood residues from timber            operations, wood shavings, and sawdust), urban wastes (e.g.            paper fraction of municipal solid waste, municipal wood            waste, municipal green waste), wood wastes (e.g. saw mill            waste, pulp mill waste, construction waste, demolition            waste, wood shavings, and sawdust), and waste paper or other            materials containing sugar, starch, and/or cellulose can be            converted to sugars and then to alcohol by fermentation with            yeast. Alternatively, materials containing sugars can be            converted directly to alcohol by fermentation.    -   Transesterification: An exemplary reaction for converting oil to        biodiesel is called transesterification. The transesterification        process reacts an alcohol (like methanol) with the triglyceride        oils contained in vegetable oils, animal fats, or recycled        greases, forming fatty acid alkyl esters (biodiesel) and        glycerin. The reaction requires heat and a strong base catalyst,        such as sodium hydroxide or potassium hydroxide.    -   Biodiesel: Biodiesel is a mixture of fatty acid alkyl esters        made from vegetable oils, animal fats or recycled greases.        Biodiesel can be used as a fuel for vehicles in its pure form,        but it is usually used as a petroleum diesel additive to reduce        levels of particulates, carbon monoxide, hydrocarbons and air        toxics from diesel-powered vehicles.    -   Hydrolysis: includes hydrolysis of a compound, e.g., a biomass,        such as a lignocellulosic material, catalyzed using an enzyme of        the instant invention.    -   Congeneration: is the simultaneous production of more than one        form of energy using a single fuel and facility. In one aspect,        biomass cogeneration has more potential growth than biomass        generation alone because cogeneration produces both heat and        electricity.

In one aspect, the polypeptides of the invention can be used inconjunction with other enzymes, e.g., hydrolases or enzymes havingcellulolytic activity, e.g., a glucanase, endoglucanase, mannase and/orother enzyme, for generating a fuel such as a bioalcohol, e.g., abioethanol, biomethanol, biobutanol or biopropanol, or biodiesel, fromany organic material, e.g., a biomass, such as compositions derived fromplants and animals, including any agricultural crop or other renewablefeedstock, an agricultural residue or an animal waste, the organiccomponents of municipal and industrial wastes, or construction ordemolition wastes or debris, or microorganisms such as algae or yeast.

In one aspect, polypeptides of the invention are used in processes forconverting lignocellulosic biomass to a fuel (e.g. a bioalcohol, e.g., abioethanol, biomethanol, biobutanol or biopropanol, or biodiesel), orotherwise are used in processes for hydrolyzing or digestingbiomaterials such that they can be used as a fuel (e.g. a bioalcohol,e.g., a bioethanol, biomethanol, biobutanol or biopropanol, orbiodiesel), or for making it easier for the biomass to be processed intoa fuel.

In an alternative aspect, polypeptides of the invention, including themixture of enzymes or “cocktails” of the invention, are used inprocesses for a transesterification process reacting an alcohol (likeethanol, propanol, butanol, propanol, methanol) with a triglyceride oilcontained in a vegetable oil, animal fat or recycled greases, formingfatty acid alkyl esters (biodiesel) and glycerin. In one aspect,biodiesel is made from soybean oil or recycled cooking oils. Animal'sfats, other vegetable oils, and other recycled oils can also be used toproduce biodiesel, depending on their costs and availability. In anotheraspect, blends of all kinds of fats and oils are used to produce abiodiesel fuel of the invention.

Enzymes of the invention, including the mixture of enzymes or“cocktails” of the invention, can also be used in glycerin refining. Theglycerin by-product contains unreacted catalyst and soaps that areneutralized with an acid. Water and alcohol are removed to produce 50%to 80% crude glycerin. The remaining contaminants include unreacted fatsand oils, which can be processes using the polypeptides of theinvention. In a large biodiesel plants of the invention, the glycerincan be further purified, e.g., to 99% or higher purity, for thepharmaceutical and cosmetic industries.

Fuels (including bioalcohols such as bioethanols, biomethanols,biobutanols or biopropanols, or biodiesels) made using the polypeptidesof the invention, including the mixture of enzymes or “cocktails” of theinvention, can be used with fuel oxygenates to improve combustioncharacteristics. Adding oxygen results in more complete combustion,which reduces carbon monoxide emissions. This is another environmentalbenefit of replacing petroleum fuels with biofuels (e.g., a fuel of theinvention). A biofuel made using the compositions and/or methods of thisinvention can be blended with gasoline to form an E10 blend (about 5% to10% ethanol and about 90% to 95% gasoline), but it can be used in higherconcentrations such as E85 or in its pure form. A biofuel made using thecompositions and/or methods of this invention can be blended withpetroleum diesel to form a B20 blend (20% biodiesel and 80% petroleumdiesel), although other blend levels can be used up to B100 (purebiodiesel).

The invention also provides processes using enzymes of this inventionfor making biofuels (including bioalcohols such as bioethanols,biomethanols, biobutanols or biopropanols, or biodiesels) fromcompositions comprising a biomass, e.g., a plant-derived source, such asa lignocellulosic biomass. The biomass material can be obtained fromagricultural crops, as a byproduct of food or feed production, or aswaste products, including lignocellulosic waste products, such as plantresidues, waste paper or construction and/or demolition wastes ordebris. Examples of suitable plant sources or plant residues fortreatment with polypeptides of the invention include kelp, algae,grains, seeds, stems, leaves, hulls, husks, corn cobs, corn stover,straw, sugar cane, sugar cane bagasse, grasses (e.g., Indian grass, suchas Sorghastrum nutans; or, switch grass, e.g., Panicum species, such asPanicum virgatum), and the like, as well as wood, wood chips, wood pulp,and sawdust. Examples of paper waste suitable for treatment withpolypeptides of the invention include discard photocopy paper, computerprinter paper, notebook paper, notepad paper, typewriter paper, and thelike, as well as newspapers, magazines, cardboard, and paper-basedpackaging materials. Examples of construction and demolition wastes anddebris include wood, wood scraps, wood shavings and sawdust.

In one embodiment, the enzymes, including the mixture of enzymes or“cocktails” of the invention, and methods of the invention can be usedin conjunction with more “traditional” means of making ethanol,methanol, propanol, butanol, propanol and/or diesel from biomass, e.g.,as methods comprising hydrolyzing lignocellulosic materials bysubjecting dried lignocellulosic material in a reactor to a catalystcomprised of a dilute solution of a strong acid and a metal salt; thiscan lower the activation energy, or the temperature, of cellulosehydrolysis to obtain higher sugar yields; see, e.g., U.S. Pat. Nos.6,660,506 and 6,423,145.

Another exemplary method that incorporates use of enzymes of theinvention, including the mixture of enzymes or “cocktails” of theinvention, comprises hydrolyzing a biomass, including anylignocellulosic material, e.g., containing hemicellulose, cellulose andlignin, or any other polysaccharide that can be hydrolyzed, bysubjecting the material to a first stage hydrolysis step in an aqueousmedium at a temperature and a pressure chosen to effect primarilydepolymerization of hemicellulose without major depolymerization ofcellulose to glucose. This step results in a slurry in which the liquidaqueous phase contains dissolved monosaccharides resulting fromdepolymerization of hemicellulose and a solid phase containing celluloseand lignin. A second stage hydrolysis step can comprise conditions suchthat at least a major portion of the cellulose is depolymerized, suchstep resulting in a liquid aqueous phase containing dissolved/solubledepolymerization products of cellulose. See, e.g., U.S. Pat. No.5,536,325. Enzymes of the invention (including the invention's mixtures,or “cocktails” of enzymes) can be added at any stage of this exemplaryprocess.

Another exemplary method that incorporated use of enzymes of theinvention, including the mixture of enzymes or “cocktails” of theinvention, comprises processing a lignocellulose-containing biomassmaterial by one or more stages of dilute acid hydrolysis with about 0.4%to 2% strong acid; and treating an unreacted solid lignocellulosiccomponent of the acid hydrolyzed biomass material by alkalinedelignification to produce precursors for biodegradable thermoplasticsand derivatives. See, e.g., U.S. Pat. No. 6,409,841. Enzymes of theinvention can be added at any stage of this exemplary process.

Another exemplary method that incorporated use of enzymes of theinvention, including the mixture of enzymes or “cocktails” of theinvention, comprises prehydrolyzing lignocellulosic material in aprehydrolysis reactor; adding an acidic liquid to the solidlignocellulosic material to make a mixture; heating the mixture toreaction temperature; maintaining reaction temperature for timesufficient to fractionate the lignocellulosic material into asolubilized portion containing at least about 20% of the lignin from thelignocellulosic material and a solid fraction containing cellulose;removing a solubilized portion from the solid fraction while at or nearreaction temperature wherein the cellulose in the solid fraction isrendered more amenable to enzymatic digestion; and recovering asolubilized portion. See, e.g., U.S. Pat. No. 5,705,369. Enzymes of theinvention can be added at any stage of this exemplary process.

The invention provides methods for making motor fuel compositions (e.g.,for spark ignition motors) based on liquid hydrocarbons blended with afuel grade alcohol made by using an enzyme or a method of the invention.In one aspect, the fuels made by use of an enzyme of the inventioncomprise, e.g., coal gas liquid- or natural gas liquid-ethanol blends.In one aspect, a co-solvent is biomass-derived 2-methyltetrahydrofuran(MTHF). See, e.g., U.S. Pat. No. 6,712,866.

In one aspect, methods of the invention for the enzymatic degradation oflignocellulose, e.g., for production of biofuels (including bioalcoholssuch as bioethanols, biomethanols, biobutanols or biopropanols, orbiodiesels) from lignocellulosic material, can also comprise use ofultrasonic treatment of the biomass material; see, e.g., U.S. Pat. No.6,333,181.

In another aspect, methods of the invention for producing biofuels(including bioalcohols such as bioethanols, biomethanols, biobutanols orbiopropanols, or biodiesels) from a cellulosic substrate compriseproviding a reaction mixture in the form of a slurry comprisingcellulosic substrate, an enzyme of this invention and a fermentationagent (e.g., within a reaction vessel, such as a semi-continuouslysolids-fed bioreactor), and the reaction mixture is reacted underconditions sufficient to initiate and maintain a fermentation reaction(as described, e.g., in U.S. Pat. App. No. 20060014260). In one aspect,experiment or theoretical calculations can determine an optimum feedingfrequency. In one aspect, additional quantities of the cellulosicsubstrate and the enzyme are provided into the reaction vessel at aninterval(s) according to the optimized feeding frequency.

One exemplary process for making biofuels (including bioalcohols such asbioethanols, biomethanols, biobutanols or biopropanols, or biodiesels)of the invention is described in U.S. Pat. App. Pub. Nos. 20050069998;20020164730; and in one aspect comprises stages of grinding thelignocellulosic biomass (e.g., to a size of 15-30 mm), subjecting theproduct obtained to steam explosion pre-treatment (e.g., at atemperature of 190-230° C.) for between 1 and 10 minutes in a reactor;collecting the pre-treated material in a cyclone or related product ofmanufacture; and separating the liquid and solid fractions by filtrationin a filter press, introducing the solid fraction in a fermentationdeposit and adding one or more enzymes of the invention, e.g., acellulase and/or beta-glucosidase enzyme (e.g., dissolved in citratebuffer pH 4.8).

Another exemplary process for making biofuels (including bioalcoholssuch as bioethanols, biomethanols, biobutanols or biopropanols, orbiodiesels) of the invention comprising bioethanols, biomethanols,biobutanols or biopropanols using enzymes of the invention comprisespretreating a starting material comprising a lignocellulosic feedstockcomprising at least hemicellulose and cellulose. In one aspect, thestarting material comprises potatoes, soybean (rapeseed), barley, rye,corn, oats, wheat, beets or sugar cane or a component or waste or foodor feed production byproduct. The starting material (“feedstock”) isreacted at conditions which disrupt the plant's fiber structure toeffect at least a partial hydrolysis of the hemicellulose and cellulose.Disruptive conditions can comprise, e.g., subjecting the startingmaterial to an average temperature of 180° C. to 270° C. at pH 0.5 to2.5 for a period of about 5 seconds to 60 minutes; or, temperature of220° C. to 270° C., at pH 0.5 to 2.5 for a period of 5 seconds to 120seconds, or equivalent. This generates a feedstock with increasedaccessibility to being digested by an enzyme, e.g., a cellulase enzymeof the invention. U.S. Pat. No. 6,090,595.

Exemplary conditions for using enzymes of the invention in thehydrolysis of lignocellulosic material include reactions at temperaturesbetween about 30° C. and 48° C., and/or a pH between about 4.0 and 6.0.Other exemplary conditions include a temperature between about 30° C.and 60° C. and a pH between about 4.0 and 8.0.

Glucanases, (or cellulases), mannanases, xylanases, amylases,xanthanases and/or glycosidases, e.g., cellobiohydrolases, mannanasesand/or beta-glucosidases can be used in the conversion of biomass tofuels, and in the production of ethanol, e.g., as described in PCTApplication Nos. WO0043496 and WO8100857. Glucanases (or cellulases),mannanases, xylanases, amylases, xanthanases and/or glycosidases, e.g.,cellobiohydrolases, mannanases and/or beta-glucosidases, can be used incombination with phytase (e.g., enzymes of the invention) to producefermentable sugars and glucan-containing biomass that can be convertedinto fuel ethanol. Amylases, glucoamylases, pullanases, glucoisomerase,alpha-glucosidase, and the like can be used in combination with phytase(e.g., enzymes of the invention) to convert starch to fermentable sugarsor ethanol. Please see PCT Application No. WO2005/096804.

Distillers Dried Grain Processing

In another aspect, the enzymes of the invention can be used totreat/process “distillers dried solubles (DDS)”, “distillers driedgrains (DDS)”, “condensed distillers solubles (CDS)”, “distillers wetgrains (DWG)”, and “distillers dried grains with solubles (DDGS)”;distillers dried grains can be a cereal byproduct of a distillationprocess, and can include solubles. These processes can comprisedry-grinding plant by-products, e.g. for feed applications, e.g., forpoultry, bovine, swine and other domestic animals. Thus, the enzymes ofthe invention can be used to treat/process grains, e.g., cereals, thatare byproducts of any distillation process, including processes usingany source of grain, for example, the traditional sources from brewers,or alternatively, from an ethanol-producing plant (factory, mill or thelike). Enzymes of the invention can be used to treat/process drying mashfrom distilleries; this mash can be subsequently used for a variety ofpurposes, e.g., as fodder for livestock, especially ruminants; thus theinvention provides methods for processing fodder for livestock such asruminants, and enzyme-processed fodder comprising phytases of thisinvention.

Phytases of this invention can be used alone or with other enzymes toprocess “distillers dried solubles (DDS)”, “distillers dried grains(DDS)”, “condensed distillers solubles (CDS)”, “distillers wet grains(DWG)”, and “distillers dried grains with solubles (DDGS)”. For example,phytases of this invention can be used in any step of an alcohol productprocess as illustrated in FIG. 10. Phytases of this invention can beused to increase the bioavailability of phosphorus in any biofuel, orpotential biofuel, including phosphorus found in “distillers driedsolubles (DDS)”, “distillers dried grains (DDS)”, “condensed distillerssolubles (CDS)”, “distillers wet grains (DWG)”, and “distillers driedgrains with solubles (DDGS)” (see, e.g., C. Martinez Amezcua, 2004Poultry Science 83:971-976).

Spirit, or Drinkable Alcohol Production

Phytases of this invention of this invention also can be used inprocessing distillers dried grains for alcohol production—alcohol as in“spirits”, e.g., beer or whiskey production (in addition to use inprocessing biomass for making biofuels). Phytases of this invention ofthis invention can be used in ethanol plants, e.g. for processing grainssuch as corn. Distillers dried grains can be made by first grinding agrain (e.g., corn) to a coarse consistency and adding to hot water.After cooling, yeast is added and the mixture ferments for several daysto a week. The solids remaining after fermentation are the distillersgrains. Phytases of this invention of this invention can be used at anystep of this process.

Formulations

The invention provides novel formulations comprising phytases, e.g., asthose described herein, and formulations for phytases, includingformulations which include the novel phytases of the invention. Thephytases of the invention can be used or formulated alone or as mixtureof phytases or phytases and other enzymes such as xylanases, cellulases,proteases, lipases, amylases, or redox enzymes such as laccases,peroxidases, catalases, oxidases, or reductases. They can be usedformulated in a solid form such as a powder, a lyophilized preparation,a granule, a tablet, a bar, a crystal, a capsule, a pill, a pellet, orin a liquid form such as in an aqueous solution, an aerosol, a gel, apaste, a slurry, an aqueous/oil emulsion, a cream, a capsule, or in avesicular or micellar suspension. The formulations of the invention cancomprise any or a combination of the following ingredients: polyols suchas a polyethylene glycol, a polyvinylalcohol, a glycerol, a sugar suchas a sucrose, a sorbitol, a trehalose, a glucose, a fructose, a maltose,a mannose, a gelling agent such as a guar gum, a carageenan, analginate, a dextrans, a cellulosic derivative, a pectin, a salt such asa sodium chloride, a sodium sulfate, an ammonium sulfate, a calciumchloride, a magnesium chloride, a zinc chloride, a zinc sulfate, a saltof a fatty acid and a fatty acid derivative, a metal chelator such as anEDTA, an EGTA, a sodium citrate, an antimicrobial agent such as a fattyacid or a fatty acid derivative, a paraben, a sorbate, a benzoate, anadditional modulating compound to block the impact of an enzyme such asa protease, a bulk proteins such as a BSA, a wheat hydrolysate, a boratecompound, an amino acid or a peptide, an appropriate pH or temperaturemodulating compound, an emulsifier such as a non-ionic and/or an ionicdetergent, a redox agent such as a cystine/cysteine, a glutathione, anoxidized glutathione, a reduced or an antioxidant compound such as anascorbic acid, a wax or oil, or a dispersant. Cross-linking and proteinmodification such as pegylation, fatty acid modification, glycosylationcan also be used to improve enzyme stability.

Measuring Metabolic Parameters

The methods of the invention involve whole cell evolution, or whole cellengineering, of a cell to develop a new cell strain having a newphenotype by modifying the genetic composition of the cell, where thegenetic composition is modified by addition to the cell of a nucleicacid of the invention. To detect the new phenotype, at least onemetabolic parameter of a modified cell is monitored in the cell in a“real time” or “on-line” time frame. In one aspect, a plurality ofcells, such as a cell culture, is monitored in “real time” or “on-line.”In one aspect, a plurality of metabolic parameters is monitored in “realtime” or “on-line.”

Metabolic flux analysis (MFA) is based on a known biochemistryframework. A linearly independent metabolic matrix is constructed basedon the law of mass conservation and on the pseudo-steady statehypothesis (PSSH) on the intracellular metabolites. In practicing themethods of the invention, metabolic networks are established, includingthe:

-   -   identity of all pathway substrates, products and intermediary        metabolites    -   identity of all the chemical reactions interconverting the        pathway metabolites, the stoichiometry of the pathway reactions,    -   identity of all the enzymes catalyzing the reactions, the enzyme        reaction kinetics,    -   the regulatory interactions between pathway components, e.g.        allosteric interactions, enzyme-enzyme interactions etc,    -   intracellular compartmentalization of enzymes or any other        supramolecular organization of the enzymes, and,    -   the presence of any concentration gradients of metabolites,        enzymes or effector molecules or diffusion barriers to their        movement.

Once the metabolic network for a given strain is built, mathematicpresentation by matrix notion can be introduced to estimate theintracellular metabolic fluxes if the on-line metabolome data isavailable.

Metabolic phenotype relies on the changes of the whole metabolic networkwithin a cell. Metabolic phenotype relies on the change of pathwayutilization with respect to environmental conditions, geneticregulation, developmental state and the genotype, etc. In one aspect ofthe methods of the invention, after the on-line MFA calculation, thedynamic behavior of the cells, their phenotype and other properties areanalyzed by investigating the pathway utilization. For example, if theglucose supply is increased and the oxygen decreased during the yeastfermentation, the utilization of respiratory pathways will be reducedand/or stopped, and the utilization of the fermentative pathways willdominate. Control of physiological state of cell cultures will becomepossible after the pathway analysis. The methods of the invention canhelp determine how to manipulate the fermentation by determining how tochange the substrate supply, temperature, use of inducers, etc. tocontrol the physiological state of cells to move along desirabledirection. In practicing the methods of the invention, the MFA resultscan also be compared with transcriptome and proteome data to designexperiments and protocols for metabolic engineering or gene shuffling,etc.

In practicing the methods of the invention, any modified or newphenotype can be conferred and detected, including new or improvedcharacteristics in the cell. Any aspect of metabolism or growth can bemonitored.

Monitoring Expression of an mRNA Transcript

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of an mRNA transcript orgenerating new transcripts in a cell. mRNA transcript, or message can bedetected and quantified by any method known in the art, including, e.g.,Northern blots, quantitative amplification reactions, hybridization toarrays, and the like. Quantitative amplification reactions include,e.g., quantitative PCR, including, e.g., quantitative reversetranscription polymerase chain reaction, or RT-PCR; quantitative realtime RT-PCR, or “real-time kinetic RT-PCR” (see, e.g., Kreuzer (2001)Br. J. Haematol. 114:313-318; Xia (2001) Transplantation 72:907-914).

In one aspect of the invention, the engineered phenotype is generated byknocking out expression of a homologous gene. The gene's coding sequenceor one or more transcriptional control elements can be knocked out,e.g., promoters or enhancers. Thus, the expression of a transcript canbe completely ablated or only decreased.

In one aspect of the invention, the engineered phenotype comprisesincreasing the expression of a homologous gene. This can be effected byknocking out of a negative control element, including a transcriptionalregulatory element acting in cis- or trans-, or, mutagenizing a positivecontrol element.

As discussed below in detail, one or more, or, all the transcripts of acell can be measured by hybridization of a sample comprising transcriptsof the cell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array.

Monitoring Expression of a Polypeptides, Peptides and Amino Acids

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of a polypeptide or generatingnew polypeptides in a cell. Polypeptides, peptides and amino acids canbe detected and quantified by any method known in the art, including,e.g., nuclear magnetic resonance (NMR), spectrophotometry, radiography(protein radiolabeling), electrophoresis, capillary electrophoresis,high performance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, various immunological methods,e.g. immunoprecipitation, immunodiffusion, immuno-electrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immuno-fluorescent assays, gel electrophoresis (e.g., SDS-PAGE),staining with antibodies, fluorescent activated cell sorter (FACS),pyrolysis mass spectrometry, Fourier-Transform Infrared Spectrometry,Raman spectrometry, GC-MS, and LC-Electrospray andcap-LC-tandem-electrospray mass spectrometries, and the like. Novelbioactivities can also be screened using methods, or variations thereof,described in U.S. Pat. No. 6,057,103. Furthermore, as discussed below indetail, one or more, or, all the polypeptides of a cell can be measuredusing a protein array.

Biosynthetically directed fractional ¹³C labeling of proteinogenic aminoacids can be monitored by feeding a mixture of uniformly ¹³C-labeled andunlabeled carbon source compounds into a bioreaction network. Analysisof the resulting labeling pattern enables both a comprehensivecharacterization of the network topology and the determination ofmetabolic flux ratios of the amino acids; see, e.g., Szyperski (1999)Metab. Eng. 1:189-197.

The following examples are intended to illustrate, but not to limit, theinvention. While the procedures described in the examples are typical ofthose that can be used to carry out certain aspects of the invention,other procedures known to those skilled in the art can also be used.

EXAMPLES Example 1: Activity Characterization of Exemplary Phytases ofthe Invention

This example describes characterizing the phytase activity ofpolypeptides of the invention, which are sequence modifications (theso-called “evolved” phytases) of the parental phytase SEQ ID NO:2, andan exemplary phytase activity assay. This phytase activity assay can beused to determine if a polypeptide has sufficient activity to be withinthe scope of the claimed invention.

After generating the polypeptides of the invention by expressing theGSSM-modified nucleic acid sequences of the invention, the “evolved”phytase polypeptides—only single residue mutation exemplary species inthis study—were purified and then heat treated (pH 7.0, 0.01% Tween) atvarious temperatures for 30 minutes. After the heat treatment step, thesamples (20 uL) were assayed with the fluorescence substrate (180 uL) 4mM DiFMUP at pH 5.5. The rates were compared to the rates of eachcorresponding non-treated sample. Results are illustrated in FIG. 1.

In another study, the “evolved” phytases of the invention comprising“blended” single mutations, i.e. phytases containing multiple mutations,were grown overnight in LBCARB100™ (LBcarb100) at 30° C. 100 uL of theeach culture (blended mutant) was heat treated on a thermocycler from72° C. to 100° C. 20 uL of the heat treated culture was mixed with 180uL of 4 mM DiFMUP at pH 5.5. The rates were compared to the rates ofeach corresponding non-treated sample; as summarized in FIG. 2. Thetable illustrated in FIG. 3 graphically summarizes the data (rounded tothe nearest tenth) used to generate the graph of FIG. 2.

Sample 1 to 21 correspond to the “blended” single mutations of theparental SEQ ID NO:2, as illustrated in the chart of FIG. 5; note that“evolved” phytase number 10 has a sequence residue modification (fromSEQ ID NO:2) that was not introduced by GSSM; this mutation wasintroduced by random chance and may or may not have any relevance tothermal stability of this exemplary phytase of the invention. Also, notethe ** marked exemplary phytases 19, 20 and 21 have C-terminal histidine(6×His) tags (—RSHHHHHH). FIG. 5 illustrates exemplary phytases havingmultiple residue modifications to the parental SEQ ID NO:2; as describedin detail herein. FIG. 6 illustrates exemplary phytases having singleresidue modifications to the parental SEQ ID NO:2; as described indetail herein.

FIG. 7 schematically illustrates an exemplary phytase assay of theinvention using the fluorescence substrate 4-methylumbelliferylphosphate (MeUMB-phosphate, whose structure is also illustrated): (i)the phytase is heat challenged for 20 minutes, 72° C., pH 4.5; and, at80° C. at physiological pH (pH 7.4); and (ii) residual activity istested at 37° C., pH 4.5:

-   -   Measure residual activity both high and low pH,    -   Calculate residual activity relative to wild-type following heat        treatment.

FIG. 8 schematically illustrates another exemplary phytase assay of theinvention that also uses the fluorescence substrate MeUMB-phosphate: (i)the phytase is heat challenged for 30 minutes, 86° C., pH 5.5; and (ii)residual activity is tested at 37° C., pH 4.5:

-   -   Measure residual activity relative to 6× variant control,    -   Select hits and re-assay at higher stringency to select top        variants.        This assay was used to screen libraries of GSSM variants (of SEQ        ID NO:1), by assaying for the phytase activity of the        polypeptides they encoded. FIG. 9 schematically illustrates the        protocol for this library screen (as described in FIG. 8), where        the library size screened is 24,576 variants.

Example 2: Development of a Phytase with Increased Thermotolerance andIncreased Gastric Lability

This example describes development and characterization of the phytaseactivity of polypeptides of the invention, which are further sequencemodifications (the “evolved” phytases) of the parental phytase SEQ IDNO:2. The evolved phytases described herein have been optimized forplant expression and for broad-acreage commercialization. The phytaseshave better or equal thermal tolerance relative to the parental phytase(SEQ ID NO:2, encoded by SEQ ID NO:1) and decreased in vitro gastricstability (increased gastric lability).

Template Selection:

The selected GSSM backbone has both thermotolerance and evidence of SGFdegradation. The evaluation began with testing the SGF properties ofthirteen thermotolerant phytase molecules (as described in Example 1,above and Table 3, below). The SGF data on the purified phytases showsthat all thermotolerant variants, including the single-sitethermotolerant parental phytase mutation N159V (SEQ ID NO:2-N159V), werevery stable, showing minimal degradation over two hours.

Previous work/literature suggest that the E. coli phytase appA gene(from strain K12, (GenBank accession no. M58708) was more susceptible toSGF degradation than the parental phytase (SEQ ID NO:2). In an effort tounderstand the SGF stability phenomenon, five of the intermediatevariants between appA and the parental phytase (SEQ ID NO:2) wereinvestigated for SGF lability. The data suggest a strong correlationbetween thermotolerance and SGF lability (FIGS. 11A and 11B). Moreimportantly, appA-7X which is one amino acid different than the parentalphytase (SEQ ID NO:2), showed ˜10% more loss in activity in SGF after 10minute incubation compared to the parental phytase (SEQ ID NO:2). Thisnew data implies that one single mutation change in SEQ ID NO:2 couldimpact changes in SGF tolerance and more importantly, be differentiatedfrom the parental phytase (SEQ ID NO:2) in the SGF assay. Based on thisnew evidence, appA-7X was selected as a benchmark control for the SGFevolution and SEQ ID NO:2 was selected as the GSSM backbone for theevolution.

Since protein purification would be an integral part of thecharacterization process, SEQ ID NO:2 was evaluated as both a his-tag(SEQ ID NO:2-HIS) and non-his tag (SEQ ID NO:2) molecule. SGF assayswere performed for both the purified his-tagged and non his-tag versionsof the parental phytase (SEQ ID NO:2). The SGF assays were performedwith two different pepsin doses (0.15 mg/mL and 0.75 mg/mL). Theresidual activity was determined at various time points (7.5, 15, 30,60, 90, and 120 minutes). There was no significant difference in SGFprofiles between the two versions (FIG. 12).

TABLE 3 Phytase variants characterized for potential GSSM SGF backbone.Parent Variant A47F W68E Q84W A95P C97E K97C T136H S168E N159V T163RD164R appA appA 2X Q84W A95P appA appA 3X Q84W A95P appA appA 4X Q84WA95P appA appA 7X W68E Q84W A95P K97C SEQ ID SEQ ID NO: 2 W68E Q84W A95PK97C S168E NO: 2 (no mutations) SEQ ID SEQ ID NO: 2- W68E Q84W A95P K97CS168E N159V NO: 2 N159V SEQ ID SEQ ID NO: 2- A47F W68E Q84W A95P K97CT136H S168E NO: 2 3X SEQ ID SEQ ID NO: 2- W68E Q84W A95P K97C T136HS168E N159V NO: 2 5Xa SEQ ID SEQ ID NO: 2- A47F W68E Q84W A95P K97CS168E N159V NO: 2 5Xb SEQ ID SEQ ID NO: 2- A47F W68E Q84W A95P K97CT136H S168E NO: 2 5Xc SEQ ID SEQ ID NO: 2- A47F W68E Q84W A95P K97CT136H S168E N159V NO: 2 5Xd SEQ ID SEQ ID NO: 2- A47F W68E Q84W A95PK97C T136H S168E N159V NO: 2 5Xe SEQ ID SEQ ID NO: 2- A47F W68E Q84WA95P K97C T136H S168E N159V NO: 2 5Xf SEQ ID SEQ ID NO: 2- A47F W68EQ84W A95P K97C T136H S168E N159V NO: 2 6X SEQ ID SEQ ID NO: 2- A47F W68EQ84W A95P K97C T136H S168E N159V D164R NO: 2 9X SEQ ID SEQ ID NO: 2-A47F W68E Q84W A95P C97E T136H S168E N159V T163R NO: 2 10Xa SEQ ID SEQID NO: 2- A47F W68E Q84W A95P K97C T136H S168E N159V T163R D164R NO: 210Xb SEQ ID SEQ ID NO: 2- A47F W68E Q84W A95P C97E K97C T136H S168EN159V T163R D164R NO: 2 13X Parent Variant E168R G179R R181Y N226C C226DV233W Q275V Y277D R289A T349Y appA appA 2X appA appA 3X Y277D appA appA4X Y277D appA appA 7X R181Y N226C Y277D SEQ ID SEQ ID NO: 2 R181Y N226CY277D NO: 2 (no mutations) SEQ ID SEQ ID NO: 2- R181Y N226C Y277D NO: 2N159V SEQ ID SEQ ID NO: 2- R181Y N226C V233W Y277D NO: 2 3X SEQ ID SEQID NO: 2- R181Y C226D V233W Y277D T349Y NO: 2 5Xa SEQ ID SEQ ID NO: 2-R181Y C226D V233W Y277D T349Y NO: 2 5Xb SEQ ID SEQ ID NO: 2- R181Y C226DV233W Y277D T349Y NO: 2 5Xc SEQ ID SEQ ID NO: 2- R181Y N226C V233W Y277DT349Y NO: 2 5Xd SEQ ID SEQ ID NO: 2- R181Y C226D Y277D T349Y NO: 2 5XeSEQ ID SEQ ID NO: 2- R181Y C226D V233W Y277D NO: 2 5Xf SEQ ID SEQ ID NO:2- R181Y C226D V233W Y277D T349Y NO: 2 6X SEQ ID SEQ ID NO: 2- G179RR181Y C226D V233W Q275V Y277D T349Y NO: 2 9X SEQ ID SEQ ID NO: 2- G179RR181Y C226D V233W Q275V Y277D T349Y NO: 2 10Xa SEQ ID SEQ ID NO: 2-G179R R181Y C226D V233W Q275V Y277D T349Y NO: 2 10Xb SEQ ID SEQ ID NO:2- E168R G179R R181Y C226D V233W Q275V Y277D R289A T349Y NO: 2 13XPurified phytase fractions of SEQ ID NO:2 and variants SEQ ID NO:2-N159Vthrough SEQ ID NO:2-6X were tested for SGF stability at pepsin doses of0.75 mg/mL and 0.15 mg/mL; data not shown. Residual activity at varioustime points (7.5, 15, 30, 60, 90, and 120 minutes) was determined. Atboth dosages, no significant difference in SGF stability between thevariant phytases was observed. SEQ ID NO:2 was the least stable in SGFat both dosages.SGF assays by SDS-PAGE analysis: All variants listed in Table 3 weretested for SGF stability by SDS-PAGE (data not shown). SDS-PAGE gelswere dyed with Simply Blue™ SafeStain. In the SGF assay, SEQ ID NO:2denatured over a 60 minute timecourse while SEQ ID NO:2-N159V and theother variants did not show significant degradation over a two hourperiod.N-Glycosylation Removal:

Previous studies suggested that N-glycosylation might improve SGFstability. Therefore, in order to reduce SFG stability, saturateddirected mutagenesis (SDM) was performed to remove the twoN-glycosylation sites on the parental SEQ ID NO:2 molecule. The firstN-glycosylation recognition site can be removed by either changing theAsparagine (N) at 161 or Threonine (T) at 163 to any other amino acid.The second N-glycosylation recognition site can be eliminated bychanging the N at 339 or the T at 341 by the same process.

SDM was conducted by constructing a primer with the desired codon changeand then utilizing PCR with the primer and the template (parentsequence) to create a new template with the desired codon change. DuringSDM all other 19 possible amino acids were substituted at each of thefour residues responsible for N-glycosylation recognition: sites 161,163, 339, and 341. The mutations showing the most similarcharacteristics (specific activity, thermotolerance, and pH profile) toSEQ ID NO:2 at the four positions were then combined to create a variantthat would not have any N-glycosylation recognition sites. The four topmutations that preserved the parental phytase's (SEQ ID NO:2) propertieswere N161K, T163R, N339E, and T341D. These mutations were combined in amanner which would remove both N-glycosylation recognition sites on thesame molecule (Table 4).

TABLE 4 N-glycosylation minus variants Name Mutations Variant GLY1 N161Kand N339E Variant GLY2 N161K and T341D Variant GLY3 T163R and N339EVariant GLY4 T163R and T341D

The four glycosylation minus variants were constructed, expressed inPichia pastoris, and characterized. The thermotolerance (½ Life) ofglycosylation-minus the variants of SEQ ID NO:2 (Variants GLY1-GLY4) andtwo SEQ ID NO:2 controls were determined by heat treatment at 80° C.over a ten minute time course. Pichia-expressed SEQ ID NO:2 andN-glycosylation-minus variants (Variants GLY1-GLY4) have approximatelythe same thermotolerance. However, SEQ ID NO:2 (expressed in Pichiapastoris) had greater thermotolerance than the same gene expressed in E.coli (SEQ ID NO:2-HIS). The lead glycosylation-minus variant, VariantGLY3, has the same thermotolerance, pH profile, and specific activity asSEQ ID NO:2 (FIG. 13). The thermotolerance data was a surprise, ahypothesis from previous work suggested that glycosylation improvedthermotolerance, therefore it was expected that the glycosylation-minusvariants would have reduced the thermotolerance. As expected, there wasa significant difference in thermotolerance between the E. coli andPichia pastoris expressed SEQ ID NO:2. However, if glycosylation is notthe factor, perhaps of the thermotolerance difference can be attributedto the different protein folding environments of the two expressionhost; Pichia—intracellular protein folding, E. coli—periplasm proteinfolding.

The T163R and N339E mutations were incorporated into the top SGF labilevariants later in the project (see TMCA^(SM) Evolution, below).

Phytase activity and pH profiles of glycosylation-minus variants(Variants GLY1-GLY4), as well as for purified Pichia expressed SEQ IDNO:2 were determined on phytate at pH 2, 2.5, 3, 4, 5, and 6 at 37° C.The data (not shown) indicates that the glycosylation-minus variantshave similar activity and pH profile to SEQ ID NO:2.

SEQ ID NO:2 SGF GSSM^(SM) Screen

SEQ ID NO:2-HIS was chosen as the GSSM template. GSSM^(SM) (Gene SiteSaturation Mutagenesis^(SM)) evolution was performed (see, for example,U.S. Pat. No. 6,171,820).

High Throughput Assay Development:

Cultures of SEQ ID NO:2 and SEQ ID NO:2-6X were grown in 384 wellmicrotiter plates were tested in an automated robotic assay format forSGF stability. The plates were heat treated at 65 C. for 30 minutes tolyse the cells. The cultures were then split into an untreated controlplate and a SGF-treated plate. The activities of the SGF-treated sampleswere compared to the untreated control samples. Time points were takenat 10, 20, 30, and 40 minutes (FIG. 14). The parental phytase's (wholecell lysate) SGF profile from the automated assay mirrored the purifiedbench scale SGF assay.

High Throughput Assay Results

The entire parental sequence (SEQ ID NO:2, except the start codon), 432codons, were mutated, expressed (in E. coli, as described below), andscreened for SGF degradation improvements utilizing the developedPhytase SGF high throughput assay. One hundred and thirty two novelmutations were confirmed for SGF lability at 69 different residuelocations (Table 5). At least eight single-site mutations met the SGFrequirement of complete protein degradation in 10 minutes. However, mostof these mutants fall short of the parental phytase's thermal toleranceproperties by 4° C. or more. Only one mutant, Q247H, had thermotoleranceequal to or slightly greater than the parental phytase and fulldegradation in SGF within 10 minutes.

SGF activity loss of select mutants from the GSSM screen was determinedover a twenty minute time course study (FIG. 15), ondifluoro-4-methylumbelliferyl phosphate (DiFMUP), 50 mM Na-Acetate, pH5.5, 0.75 mg/mL pepsin. The characterization of these SGF mutationsshowed that the rate for complete degradation fell into threecategories, fast (less than 2 minutes), medium (˜10-15 minutes), andslow (≧20 minutes); slow SGF mutants showed just a slight improvementover the parental phytase (SEQ ID NO:2). The data suggest that theactivity analysis is a more sensitive method to determine phytase decay;at 20% residual activity the phytase band is not detectable on theSDS-Page gel (data not shown).

The SGF mutants were tested for thermotolerance by two methods; a) 65°C. thirty minute heat treatment and b) thirty minute 50-70° C. gradient.The thermotolerance data suggest that most of the SGF mutations lost ≧4°C. with the exception of variant I427T and variant Q247H. To quicklyovercome the thermotolerance deficiencies, a fast track strategy wasdeveloped; several very promising SGF mutations were incorporated intomore thermotolerant phytase backbones (from Example 1, above) to test ifthermotolerance can be regained (discussed in depth under title FastTrack Strategy).

All mutations were compared and the top 48 were ranked (Table 6) by aFitness Value FT (Thermotolerance %−SGF %=FT). The top single-sitemutations were considered for combination in the Tailored Multi-SiteCombinatorial Assembly (TMCA^(SM)) phase (see below).

Based on the data, there were certain amino acid substitutions andresidue positions, when changed in the parental phytase (SEQ ID NO:2)molecule, which were more favorable for SGF lability improvements thanothers (FIG. 16). In FIG. 16, the number below the amino acid symbolindicates how many times the molecule is represented in the originalprotein. For example, residue position 48, a Threonine, could be changedto nine other amino acids (FYWMHKVIL). The three amino acid additionsthat most frequently increased SGF lability were Leucine (14 times),Proline (12 times), and Histidine (12 times). As a further example,Arginines which were in the original protein (22 times in SEQ ID NO:2)were never replaced with another amino acid where an accompanyingincrease in SFG lability was observed. However, when Arginine wassubstituted for another amino acid in the original protein (7 times), ineach instance, SGF lability was increased. The SGF GSSM data suggestedthat hot spots for SGF mutations occurred in the phytase molecule (FIG.17). The largest series of mutations, seven in row, occurred betweenresidues 145-151. There were also three sets where three residues in arow could be mutated. Several amino acid residues were promiscuous toamino acid substitutions favoring SGF lability, the most extreme beingT48, which was replaced by nine different amino acids (F, Y, W, M, H, K,V, I, and L). For position 48 and 79, H was the best mutation andselected as a candidate for the TMCA library.

TABLE 5 Variant names and mutations of the hits discovered from the GSSMscreen which have SGF degradation improvements. Variant Mutation 1 P 100A 2 P 149 L 3 I 427 T 4 T 291 W 5 T 291 V 6 L 126 R 7 P 254 S 8 L 192 F9 Q 377 R 10 V 422 M 11 L 157 P 12 I 107 H 13 I 108 R 14 Q 309 P 15 I108 A 16 I 108 S 17 I 107 P 18 C 155 Y 19 I 108 Q 20 A 236 T 21 S 208 P22 A 109 V 23 G 171 M 24 S 173 G 25 V 162 L 26 D 139 Y 27 L 146 R 28 Q137 Y 29 Q 137 L 30 L 146 T 31 K 151 P 32 N 148 K 33 K 151 H 34 Q 137 F35 L 157 C 36 L 150 Y 37 V 162 T 38 I 174 P 39 G 353 C 40 L 150 T 41 S102 A 42 I 174 F 43 G 171 S 44 N 148 M 45 Q 137 V 46 P 145 L 47 I 108 Y48 E 113 P 49 F 147 Y 50 S 173 H 51 T 163 P 52 N 148 R 53 S 173 V 54 A248 L 55 A 248 T 56 Q 247 H 57 A 236 H 58 L 269 I 59 S 197 G 60 L 235 I61 S 211 H 62 T 282 H 63 Q 246 W 64 G 257 R 65 L 269 T 66 G 257 A 67 F194 L 68 H 272 W 69 V 191 A 70 S 218 Y 71 P 217 S 72 P 217 D 73 P 217 G74 S 102 Y 75 S 218 I 76 A 232 P 77 W 265 L 78 N 266 P 79 L 167 S 80 L216 T 81 P 217 L 82 L 244 S 83 P 269 L 84 T 48 F 85 T 48 W 86 T 48 M 87T 48 H 88 T 48 K 89 T 48 Y 90 T 48 V 91 M 51 A 92 M 51 L 93 T 48 I 94 M51 G 95 T 48 L 96 L 50 W 97 G 67 A 98 Y 79 W 99 Y 79 N 100 P 149 N 101 Y79 H 102 Q 86 H 103 Q 275 H 104 A 274 I 105 A 274 T 106 Y 79 S 107 H 263P 108 A 274 V 109 A 274 L 110 A 274 F 111 S 389 V 112 G 395 T 113 G 395Q 114 G 395 L 115 G 395 I 116 G 395 E 117 S 389 H 118 I 427 G 119 I 427S 120 A 429 P 121 P 343 E 122 P 343 V 123 P 343 R 124 P 343 L 125 P 343V 126 N 348 K 127 P 343 I 128 N 348 W 129 P 343 N 130 L 379 V 131 Q 381S 132 L 379 SA significant number of the GSSM variants met the SGF requirements (FastDegradation<10% after 10 Min SGF survival), but are short on thethermotolerance properties (<75% survival after 65° C. 30 min heattreatment). The Medium and Slow degrading mutants met thethermotolerance requirements, but not SGF requirements. From the GSSMscreen, only Variant 56 (Q247H) met both SGF and Thermotoleranceproperties.

TABLE 6 Ranking Top SGF mutants. Fitness Rank Mutation 65 C HT % SGF %Value 1 Q247H 76 3 0.73 2 I427T 99 37 0.62 3 Q246W 76 23 0.53 4 L157P 462 0.44 5 Q377R 47 3 0.44 6 T48M 66 24 0.42 7 A274V 60 17 0.42 8 A236T 475 0.42 9 Q275H 77 35 0.41 10 T48W 56 15 0.41 11 I174P 44 4 0.41 12 T48H50 10 0.40 13 Y79H 67 27 0.40 14 A232P 41 1 0.40 15 T48K 53 13 0.40 16T48Y 63 24 0.39 17 ? 45 7 0.38 18 P217D 40 3 0.37 19 P217G 43 7 0.37 20P217S 42 5 0.37 21 T48I 70 34 0.36 22 P343V 41 5 0.36 23 S211H 67 310.35 24 T291V 36 2 0.34 25 A274I 51 16 0.34 26 L50W 58 26 0.33 27 P343E40 7 0.33 28 M51L 60 28 0.33 29 G257A 36 3 0.32 30 H263P 60 29 0.32 31Y79S 67 36 0.31 32 Y79N 68 38 0.30 33 T48F 50 21 0.29 34 L296T 62 340.29 35 S218Y 30 2 0.28 36 P343R 32 6 0.26 37 T48L 48 24 0.24 38 P149L36 12 0.24 39 L167S 42 18 0.23 40 G67A 59 36 0.23 41 P343N 47 25 0.22 42P343L 26 6 0.20 43 A236H 19 0 0.19 44 ? 36 20 0.15 45 T291W 18 3 0.14 46SEQ ID NO: 2 74 64 0.10 47 SEQ ID NO: 2 70 61 0.09 48 S208P 11 2 0.08 49L192F 22 13 0.08 50 SEQ ID NO: 2 64 57 0.07 51 SEQ ID NO: 2 66 59 0.0752 SEQ ID NO: 2 63 56 0.07 53 SEQ ID NO: 2 67 62 0.50 54 Q377R 7 3 0.0455 Q309P 1 2 −0.01 SEQ ID NO: 2 67 60 0.07 AVERAGE All 132 mutants werecompared side by side to determine the best mutants from the SGF screen.The top 49 SGF mutants ranked and compared with six separate controls(parental phytase - SEQ ID NO: 2). Variants were ranked on theirthermotolerance properties (% survival at 65° C. HT %) and SGF lability(SGF survival - SGF %). An arbitrary Fitness Value (FV) = (65° C. HT %)− (SGF %) was established and the variants with the higher FV,highlighted in orange, were considered for further evolution using theTMCA technology.Two of the top three mutations (Q246W and Q247H) showed thermotoleranceproperties similar to the parental phytase (SEQ ID NO:2) and significantimprovements in SGF lability. Using 3-D modeling, it was observed thatW246 is predicted to be buried beneath the surface of the protein,necessitating the protein to adapt to a larger tryptophan side chain inwhat was a tightly packed environment around a glutamine side chain.Also, the protein must adapt to the loss of the hydrogen bond betweenthe epsilon oxygen of the Q246 side chain with the main chain nitrogenof G255 as there is no side chain oxygen in tryptophan residues.Structural analysis also indicated that while the main chain of H247would be buried, the imidizole side chain would snorkel to the surface.Although glutamine to histidine is a relatively conservative change,this creates a new surface accessible group in this region that isavailable for protonation at lower pH which would clearly alter thelocal hydrogen bonding network, potentially acting as a acidic switchfor disrupting the local protein structure in this region leaving theprotein more susceptible to acid and pepsin degradation.Fast Track Strategy

To overcome the thermotolerance properties which were lost in the leadSGF labile variants (except Q247H), a Fast Track Strategy was initiatedto design phytase molecules which have both the desired properties; SGFand thermotolerance. Thermotolerant phytases (SEQ ID NO:2-N159V, SEQ IDNO:2-6X and SEQ ID NO:2-9×) from the earlier evolution work (see Example1, above) were selected as the backbones for two rounds of Site DirectedMutagenesis (SDM).

The first SDM incorporated each of the single SGF mutations (T291V,A236T, or L157P) into each of the three thermotolerant backbones asshown in the table below (Table 7).

TABLE 7 SDM Round I variants SDM round I Host- His TOP AdditionalVariant Parent (backbone) Tag 10 Mutations A SEQ ID NO: 2-6X x x T291V BSEQ ID NO: 2-6X x x A236T C SEQ ID NO: 2-6X x x L157P D SEQ ID NO:2-N159V x x L157P E SEQ ID NO: 2-N159V x x A236T F SEQ ID NO: 2-N159V xx T291V G SEQ ID NO: 2-9X x x L157P H SEQ ID NO: 2-9X x x A236T I SEQ IDNO: 2-9X x x T291V

The data from this first round of SDM suggested that improvements in SGFlability was made to the variants made from backbone SEQ ID NO:2-N159V.However, those SGF mutations also decreased the thermotoleranceproperties below the objective. The variants of the other twothermotolerant backbones (SEQ ID NO:2-6X and SEQ ID NO:2-9X) showed onlyminimal improvements in SGF degradation. It was also observed thatadding more SGF mutations to the SEQ ID NO:2-N159V backbone reduced thethermotolerance below the thermotolerance objective.

A second round of SDM adding more SGF mutations to the other twothermotolerant variants was performed by incorporating up to two SGFmutations (T291V and/or L192F) into SEQ ID NO:2-6X and SEQ ID NO:2-9X,both with or without the A236T mutation (see Table 8 below).

TABLE 8 SDM Round II variants SDM round II Host- His TOP AdditionalVariant Parent (backbone) Tag 10 Mutations J SEQ ID NO: 2-6X x x L192F KSEQ ID NO: 2-6X x x T291V L SEQ ID NO: 2-6X x x T291V + L192F M SEQ IDNO: 2-9X x x L192F N SEQ ID NO: 2-9X x x T291V O SEQ ID NO: 2-9X x xT291V + L192F P SEQ ID NO: 2-6X + (A236T) x x L192F Q SEQ ID NO: 2-6X +(A236T) x x T291V R SEQ ID NO: 2-6X + (A236T) x x T291V + L192F S SEQ IDNO: 2-9X + (A236T) x x L192F T SEQ ID NO: 2-9X + (A236T) x x T291V U SEQID NO: 2-9X + (A236T) x x T291V + L192F

The result from this SDM produced a variant, Variant O, with slightlybetter thermal tolerance than the parental phytase (SEQ ID NO:2) andfull degradation in less than 10 min in SGF. For example, at T-0 andT-10 (min) time points using different SGF pepsin dosages (0.3, 0.2,0.1, 0.05, 0.025 and 0 mg/mL pepsin), SDS-PAGE gels dyed with SimplyBlue™ SafeStain, showed that Variant O was fully degraded within 10minutes at 0.3 mg/mL and 0.2 mg/mL pepsin. A twenty minute SGF timecourse (at 0.3 mg/mL pepsin), again run on SDS-PAGE gels, showed thatSEQ ID NO:2-HIS is not degraded after 20 minutes, while Variant O isfully degraded around 7.5 minutes.

SGF stability of Variant O and SEQ ID NO:2-HIS was determined atdifferent pepsin dosages: 0.3, 0.2, 0.1, 0.05, 0.025, and 0.0 mg/mLPepsin (FIG. 18). In FIG. 18, the 0.3 mg/mL pepsin dose for SEQ IDNO:2-HIS is graphed as a benchmark. The dosage response experimentsindicated that pepsin is required for complete degradation and that itis not just a function of the acidic treatment (FIG. 18).

The ½ Life of Variant O and SEQ ID NO:2-HIS was determined at 75° C.(FIG. 19). Purified parental phytase (SEQ ID NO:2-HIS) and phytasevariant Variant O, in two different buffers (100 mM Citrate pH 5.5 and100 mM Tris pH 7.2), were heat treated at 75 C.° up to 45 minutes (T-0,5, 10, 15, 20, 30, 45 min). Ten microliters of the heat treated sampleswere assayed for activity in 100 μl, 100 μM DiFMUP, 50 mM Citrate pH5.5. Activity was compared to T-0 activity. Variant O met the SGF andthermotolerance requirements, however, specific activity was lower thandesired (FIG. 19). Loss of specific activity was expected because ofprevious knowledge from the earlier evolution work (Example 1)indicating that the SEQ ID NO:2-6X backbone only had ⅔ of the specificactivity of the parental phytase (SEQ ID NO:2).

To maximize the TMCA strategy, and overcome Variant O deficiencies, theapproach was to blend thermotolerant mutations that maintained specificactivity with SGF mutations using the parental phytase (SEQ ID NO:2-HIS)as the template.

TMCA^(SM) Evolution

The high throughput assay was modified to include a heat treatment stepin order to select for variants that desired thermotolerant as well asthe SGF properties. Two libraries were created utilizing the TailoredMulti-Site Combinatorial Assembly (TMCA) technology (see PCT PublicationNo. WO 09/018,449). TMCA evolution comprises a method for producing aplurality of progeny polynucleotides having different combinations ofvarious mutations at multiple sites. The method can be performed, inpart, by a combination of at least one or more of the following steps:

Obtaining Sequence Information of a (“First” or “Template”)Polynucleotide.

For example, the first or template sequence can be a wild type (e.g. SEQID NO:2-N159V) or mutated (e.g. the “D164R template”, described below)sequence. The sequence information can be of the complete polynucleotide(e.g., a gene or an open reading frame) or of partial regions ofinterest, such as a sequence encoding a site for binding,binding-specificity, catalysis, or substrate-specificity.

Identifying Three or More Mutations of Interest Along the First orTemplate Polynucleotide Sequence.

For example, mutations can be at 3, 4, 5, 6, 8, 10, 12, 20 or morepositions within the first or template sequence. The positions can bepredetermined by absolute position or by the context of surroundingresidues or homology. For TMCA of phytase polypeptides, the top SGF andthermotolerant amino acid changes that resulted in improved enzymeperformance were included as mutations of interest. The sequencesflanking the mutation positions on either side can be known. Eachmutation position may contain two or more mutations, such as fordifferent amino acids. Such mutations can be identified by using GeneSite Saturation Mutagenesis^(SM) (GSSM^(SM)) technology, as describedherein and in U.S. Pat. Nos. 6,171,820; 6,562,594; and 6,764,835.

Providing Primers (e.g., Synthetic Oligonucleotides) Comprising theMutations of Interest.

In one embodiment, a primer is provided for each mutation of interest.Thus, a first or template polynucleotide having 3 mutations of interestcan use 3 primers at that position. The primer also can be provided as apool of primers containing a degenerate position so that the mutation ofinterest is the range of any nucleotide or naturally occurring aminoacid, or a subset of that range. For example, a pool of primers can beprovided that favor mutations for aliphatic amino acid residues.

The primers can be prepared as forward or reverse primers, or theprimers can be prepared as at least one forward primer and at least onereverse primer. When mutations are positioned closely together, it canbe convenient to use primers that contain mutations for more than oneposition or different combinations of mutations at multiple positions.

Providing a Polynucleotide Containing the Template Sequence.

The first or template polynucleotide can be circular, or can besupercoiled, such as a plasmid or vector for cloning, sequencing orexpression. The polynucleotide may be single-stranded (“ssDNA”), or canbe double-stranded (“dsDNA”). For example, the TCMA method subjects thesupercoiled (“sc”) dsDNA template to a heating step at 95° C. for 1 min(see Levy, Nucleic Acid Res., 28(12):e57(1-vii) (2000)).

Adding the Primers to the Template Polynucleotide in a Reaction Mixture.

The primers and the template polynucleotide are combined underconditions that allow the primers to anneal to the templatepolynucleotide. In one embodiment of the TMCA protocol, the primers areadded to the polynucleotide in a single reaction mixture, but can beadded in multiple reactions.

Performing a Polymerase Extension Reactions.

The extension products (e.g., as a “progeny” or “modified extendedpolynucleotide”) may be amplified by conventional means. The productsmay be analyzed for length, sequence, desired nucleic acid properties,or expressed as polypeptides. Other analysis methods include in-situhybridization, sequence screening or expression screening. The analysiscan include one or more rounds of screening and selecting for a desiredproperty.

The products can also be transformed into a cell or other expressionsystem, such as a cell-free system. The cell-free system may containenzymes related to DNA replication, repair, recombination,transcription, or for translation. Exemplary hosts include bacterial,yeast, plant and animal cells and cell lines, and include E. coli,Pseudomonas fluorescens, Pichia pastoris and Aspergillus niger. Forexample, XL1-Blue or Stb12 strains of E. coli can be used as hosts.

The method of the invention may be used with the same or differentprimers under different reaction conditions to promote products havingdifferent combinations or numbers of mutations.

By performing the exemplary method described above, this protocol alsoprovides one or more polynucleotides produced by this TMCA evolutionmethod, which then can be screened or selected for a desired property.One or more of the progeny polynucleotides can be expressed aspolypeptides, and optionally screened or selected for a desiredproperty. Thus, this embodiment of the TMCA evolution protocol providespolynucleotides and the encoded polypeptides, as well as libraries ofsuch polynucleotides encoding such polypeptides. This embodiment of theTMCA evolution protocol further provides for screening the libraries byscreening or selecting the library to obtain one or more polynucleotidesencoding one or more polypeptides having the desired activity.

Another embodiment of the TMCA evolution protocol described in PCTPublication No. WO 2009/018449 comprises a method of producing aplurality of modified polynucleotides. Such methods generally include(a) adding at least three primers to a double stranded templatepolynucleotide in a single reaction mixture, wherein the at least threeprimers are not overlapping, and wherein each of the at least threeprimers comprise at least one mutation different from the other primers,wherein at least one primer is a forward primer that can anneal to aminus strand of the template and at least one primer is a reverse primerthat can anneal to a plus strand of the template, and (b) subjecting thereaction mixture to a polymerase extension reaction to yield a pluralityof extended modified polynucleotides from the at least three primers.

Another embodiment of the TMCA evolution protocol described in PCTPublication No. WO 2009/018449 comprises a method wherein a cell istransformed with the plurality of extended products that have not beentreated with a ligase. In another embodiment of the invention, theplurality of extended modified polynucleotides is recovered from thecell. In another embodiment, the recovered plurality of extendedmodified polynucleotides is analyzed, for example, by expressing atleast one of the plurality of extended modified polynucleotides andanalyzing the polypeptide expressed therefrom. In another embodiment,the plurality of extended modified polynucleotides comprising themutations of interest is selected.

In another embodiment of the TMCA evolution protocol, sequenceinformation regarding the template polynucleotide is obtained, and threeor more mutations of interest along the template polynucleotide can beidentified. In another embodiment, products obtained by the polymeraseextension can be analyzed before transforming the plurality of extendedmodified products into a cell.

In one embodiment of the TMCA evolution protocol, products obtained bythe polymerase extension are treated with an enzyme, e.g., a restrictionenzyme, such as a DpnI restriction enzyme, thereby destroying thetemplate polynucleotide sequence. The treated products can betransformed into a cell, e.g., an E. coli cell.

In one embodiment of the TMCA evolution protocol, at least two, or atleast three, or at least four, or at least five, or at least six, or atleast seven, or at least eight, or at least nine, or at least ten, or atleast eleven, or at least twelve, or more primers can be used. In oneembodiment, each primer comprises a single point mutation. In anotherembodiment, two forward or two reverse primers comprise a differentchange in the same position on the template polynucleotide. In anotherembodiment, at least one primer comprises at least two changes indifferent positions on the template polynucleotide. In yet anotherembodiment, at least one primer comprises at least two changes indifferent positions and at least two forward or two reverse primerscomprise a different change in the same position on the templatepolynucleotide.

In one embodiment of the TMCA evolution protocol, the forward primersare grouped into a forward group and the reverse primers are groupedinto a reverse group, and the primers in the forward group and theprimers in the reverse group, independent of one another, are normalizedto be equal concentration in the corresponding group regardless ofpositions on the template polynucleotide, and wherein after thenormalization an equal amount of the forward and reverse primers isadded to the reaction. In this normalization method, a combination ofsome positions may be biased. The bias can be due to, for example, arelatively low primer concentration at one position containing a singleprimer compared to a position containing multiple primers. “Positionalbias” refers to resulting polynucleotides which show a strong preferencefor the incorporation of primers at a single position relative to theother positions within its forward or reverse primer group. This resultsin a combination of modified polynucleotides which may have a highpercentage of mutations within a single primer position but a lowpercentage of mutations at another position within its forward orreverse primer group. This bias is unfavorable when the goal of the TMCAis to generate progeny polynucleotides comprising all possiblecombinations of changes to the template. The bias can be corrected, forexample, by normalizing the primers as a pool at each position to beequal.

In one embodiment of the TMCA evolution protocol, the primernormalization is performed by organizing the primers into multiplegroups depending on their location on the template polynucleotide,wherein the primers covering the same selected region on the templateare in one group; normalizing the grouped primers within each group tobe equal concentration; pooling the forward primers within one groupinto a forward group and normalizing concentration between each group ofthe forward primers to be equal; pooling the reverse primers within onegroup into a reverse group and normalizing concentration between eachgroup of the reverse primers to be equal; and adding an equal amount ofthe pooled forward and reversed primers into the reaction. No bias hasbeen observed for position combinations.

In one embodiment of the TMCA evolution protocol, a set of degenerateprimers each comprising a degenerate position is provided, wherein themutation of interest is a range of different nucleotides at thedegenerate position. In another embodiment, a set of degenerate primersis provided comprising at least one degenerate codon corresponding to atleast one codon of the template polynucleotide and at least one adjacentsequence that is homologous to a sequence adjacent to the codon of thetemplate polynucleotide sequence. In another embodiment, the degeneratedcodon is N,N,N and encodes any of 20 naturally occurring amino acids. Inanother embodiment, the degenerated codon encodes less than 20 naturallyoccurring amino acids.

Another embodiment of the TMCA evolution protocol described in PCTPublication No. WO 2009/018449 comprises a method of producing aplurality of modified polynucleotides comprising the mutations ofinterest. Such methods generally include (a) adding at least two primersto a double stranded template polynucleotide in a single reactionmixture, wherein the at least two primers are not overlapping, andwherein each of the at least two primers comprise at least one mutationdifferent from the other primer(s), wherein at least one primer is aforward primer that can anneal to a minus strand of the template and atleast one primer is a reverse primer that can anneal to a plus strand ofthe template, (b) subjecting the reaction mixture to a polymeraseextension reaction to yield a plurality of extended modifiedpolynucleotides from the at least two primers, (c) treating theplurality of extended modified polynucleotides with an enzyme, therebydestroying the template polynucleotide, (d) transforming the treatedextended modified polynucleotides that have not been treated with aligase into a cell, (e) recovering the plurality of extended modifiedpolynucleotides from the cell, and (f) selecting the plurality ofextended modified polynucleotides comprising the mutations of interest.

Using the TMCA technology, a small library, Library A, was created forquick turn around and to simplify the process (96 different variantscontaining up to five SGF mutations and two thermotolerance mutations,see Table 9). Library A used a single template, SEQ ID NO:2-N159V andthe oligoes listed in Table 10, below. A second more extensive library,Library B, was also created using TMCA technology in order to increasethe potential of creating a very thermotolerant variant with therequired SGF properties (4096 different variants containing up to sevenSGF mutations and five thermotolerance mutations, see Table 9). LibraryB used two templates, SEQ ID NO:2-N159V and “D164R template”, in twoseparate TMCA reactions each using the oligoes listed in Table 11,creating two sub-libraries. The “D164R template” was generated inLibrary A and consisted of the SEQ ID NO:2-N159V backbone with the D164Rmutation incorporated. Both libraries were amplified into the pQE60vector (Qiagen, Valencia, Calif.) and then transformed into host PHY635(described below) to confirm primary and secondary phytase activity.

A total of eight promising thermotolerant SGF labile hits werediscovered by screening the libraries. Five thermotolerant (Tm˜5° C.greater than the parental phytase, SEQ ID NO:2, Table 13) SGF labilevariants were discovered in the small library (Variants AA-EE, Table12). The larger TMCA library produced ten candidates, however, onlythree had greater thermotolerance than Variants AA-EE (Variants FF-HH,Table 13, had Tm 7.5° C. greater than the parental phytase, SEQ IDNO:2). For characterization screening, these variants, along with thebest single-site SGF mutation (Variant 56), all as glycosylated andglycosylation minus versions, were expressed in Pichia pastoris,(glycosylation minus versions included the two mutations, T163R andN339E, from the N-glycosylation removal research, see above).

Residual activity of SGF labile phytase variants during SGF treatmentwas determined (FIG. 20). Purified parental phytase (SEQ ID NO:2),Variant 56 and Variant AA-HH were treated with SGF (pH 1.2) with pepsin(10 U/μg phytase) over a 10.0 minute time course. Phytase stability wasdetermined by activity on DiFMUP. The specific activity of SGF labilephytase variants compared to SEQ ID NO:2 phytase (FIG. 21). Purifiedparental phytase (SEQ ID NO:2) and lead phytase variants were tested foractivity on phytate. Purified protein was assayed in 4 mM phytate, 100mM Na-Acetate pH 4.5 at 37° C. There was not any significant change inSGF and thermotolerance properties between the glycosylated andnon-glycosylated Pichia expressed lead variants (FIGS. 20 and 21).Previous work predicted that the glycosylated variants would have ahigher thermotolerance and be more tolerant to SGF; our data suggestedotherwise.

Also, a pH profile of glycosylated, glycosylation-minus variants, andthe parental phytase (SEQ ID NO:2) was generated for phytase activity onphytate at pH 2, 2.5, 3, 4, 5, and 6 at 37° C. All phytases assayed hadvery similar pH profiles (data not shown).

TABLE 9 Mutations selected for TMCA evolution. Library A Library BThermotolerant Thermotolerant SGF Mutations Mutations SGF MutationsMutations Q247H Q275V Q247H G179R I427T D164R I427T Q275V L157P L157PT349Y Q275H Q275H C226D T48M T48M D164R Q246W Q377R Y79H Thermotolerancemutations and SGF mutations were blended utilizing TMCA technology,using SEQ ID NO: 2-N159V as the backbone.

TABE 10 Oligoes used in Library A Oligo name Oligo sequence - 5′-3′T48M_F TGCGTGCTCCAACCAAGGCCATGCAACTGATGCAGGATGTCA SEQ ID NO: 3 C L157P_FTAAAAACTGGCGTTTGCCAACCGGATGTGGCGAACGTGACTG SEQ ID NO: 4ACGCGATCCTCGAGAGGGCAGGA D164R_FTAAAAACTGGCGTTTGCCAACTGGATGTGGCGAACGTGACTC SEQ ID NO: 5GTGCGATCCTCGAGAGGGCAGGA L157P-TAAAAACTGGCGTTTGCCAACCGGATGTGGCGAACGTGACTC SEQ ID NO: 6 D164_RGTGCGATCCTCGAGAGGGCAGGA Q247H_RGAGATATTTCTCCTGCAACATGCACAGGGAATGCCGGAGCC SEQ ID NO: 7 Q275H_RTGCTAAGTTTGCATAACGCGCATTTTGATTTGCTACAACGCAC SEQ ID NO: 8 Q275V_RTGCTAAGTTTGCATAACGCGGTGTTTGATTTGCTACAACGCAC SEQ ID NO: 9 I427T_RAATCGTGAATGAAGCACGCACACCGGCGTGCAGTTTGAGAT SEQ ID NO: 10

TABLE 11 Oligoes used in Library B Oligo name Oligo sequence - 5′-3′T48M_F TGCGTGCTCCAACCAAGGCCATGCAACTGATGCAGGA SEQ ID NO: 11 TGTCAC Y79H_FGCGGTGGTGAGCTAATCGCCCATCTCGGACATTACTG SEQ ID NO: 12 GCGTCA L157P_FTAAAAACTGGCGTTTGCCAACCGGATGTGGCGAACGT SEQ ID NO: 13 GACTGA G179R_FGGTCAATTGCTGACTTTACCCGCCATTATCAAACGGCG SEQ ID NO: 14 TTTCG C226D_FAACTCAAGGTGAGCGCCGACGATGTCTCATTAACCGG SEQ ID NO: 15 TGCGGT Q246W_RTGACGGAGATATTTCTCCTGTGGCAAGCACAGGGAAT SEQ ID NO: 16 GCCGGA Q246W +Q247H_R TGACGGAGATATTTCTCCTGTGGCATGCACAGGGAAT SEQ ID NO: 17 GCCGGAGCCQ247H_R CGGAGATATTTCTCCTGCAACATGCACAGGGAATGCC SEQ ID NO: 18 GGAGCCQ275V_R TGCTAAGTTTGCATAACGCGGTGTTTGATTTGCTACAA SEQ ID NO: 19 CGCACT349Y_R TTCCCGGTCAGCCGGATAACTATCCGCCAGGTGGTGA SEQ ID NO: 20 ACTGGTQ377R_R TTCAGGTTTCGCTGGTCTTCCGCACTTTACAGCAGATG SEQ ID NO: 21 CGTGAI427T_R AATCGTGAATGAAGCACGCACACCGGCGTGCAGTTTG SEQ ID NO: 22 AGAT

TABLE 12 Sequence of Lead SGF Labile Thermotolerant Phytase variantsType of mutation SGF SGF SGF Thermo Glycos Thermo Thermo Thermo SGFVariant T48M Y79H L157P N159V T163R D164R G179R C226D Q246W AA X X X BBX X X CC X X X X DD X X X X EE X X X FF X X X X X GG X X X X X X X HH XX X X X X X 56 X Type of mutation SGF Thermo SGF Glycos Thermo SGF SGFVariant Q247H Q275V Q275H N339E T349Y Q377R I427T AA X X X BB X X X CC XX X X DD X X EE X X X FF X X X X GG X X X HH X X X 56 X XVariant AA is SEQ ID NO:28 (encoded by SEQ ID NO:27), Variant BB is SEQID NO:32 (encoded by SEQ ID NO:31), Variant CC is SEQ ID NO:34 (encodedby SEQ ID NO:33), Variant DD is SEQ ID NO:36 (encoded by SEQ ID NO:35),Variant EE is SEQ ID NO:38 (encoded by SEQ ID NO:37), Variant FF is SEQID NO:24 (encoded by SEQ ID NO:23), Variant GG is SEQ ID NO:26 (encodedby SEQ ID NO:25), Variant HH is SEQ ID NO:40 (encoded by SEQ ID NO:39),and Variant 56 is SEQ ID NO:30 (encoded by SEQ ID NO:29). Note, however,that SEQ ID NOs:23, 25, 27, 29, 31, 33, 35, 37, and 39, do not includethe nucleic acids encoding the native signal sequence and that SEQ IDNOs:24, 26, 28, 30, 32, 34, 36, 38, and 40, do include the native signalsequence amino acids (amino acids 1-22 of SEQ ID NO:2). A startMethionine (ATG) is added in each of the referenced sequences. Thepositions of the point mutations for these variants (listed e.g. inTable 12) are counted as if the native signal sequence is present.

TABLE 13 Melting Temperature (Tm) of SEQ ID NO: 2 and Lead SGFCandidates. Glycosylation Glycosylated Minus DSC Tm Library (Tm inCelsius) (Tm in Celsius) SEQ ID NO: 2 79.8 N/A Variant AA (SEQ ID NO:28) A 85.2 85.8 Variant BB (SEQ ID NO: 32) A 84.2 85.8 Variant CC (SEQID NO: 34) A 85.6 85.2 Variant DD (SEQ ID NO: 36) A 83.8 84.2 Variant EE(SEQ ID NO: 38) A 85.4 84.6 Variant FF (SEQ ID NO: 24) B 87.5 87.6Variant GG (SEQ ID NO: 26) B 87.3 87.0 Variant HH (SEQ ID NO: 40) B 87.486.2 Variant 56 (SEQ ID NO: 30) 81.0 81.3 Purified parental phytase (SEQID NO: 2) and the nine lead SGF phytase candidates were expressed inPichia pastoris, purified, dialyzed in 100 mM Citrate pH 5.5, and testedfor Tm utilizing the Applied Thermodynamics N-DSCII.Selection of Top Four Variants for Animal Studies

-   -   SGF SDS-PAGE analysis at nine time points (0, 0.5, 1.0, 1.5,        2.0, 3.0, 4.0, 5.0, and 10 minutes), with pepsin dosage of 10 U        per μg phytase, four variants were selected based on this        characterization data (not shown) for large scale fermentation        to be used in animal trials. The four selected leads showed        complete protein degradation within five minutes.

Selected Leads:

-   -   Variant 56 (SEQ ID NO:30, encoded by SEQ ID NO:29) is the        closest variant to the original parental phytase (SEQ ID NO:2)        molecule, having one SGF mutation and two glycosylation-minus        mutations.    -   Variant AA (SEQ ID NO:28, encoded by SEQ ID NO:27) has two SGF        mutations, two thermotolerant mutations and two        glycosylation-minus mutations.    -   Variant FF (SEQ ID NO:24, encoded by SEQ ID NO:23) has three SGF        mutations, four thermotolerant mutations and two        glycosylation-minus mutations.    -   Variant GG (SEQ ID NO:26, encoded by SEQ ID NO:25) has three SGF        mutations, five thermotolerant mutations, two        glycosylation-minus mutations.        Fermentation of Lead Candidates:

The leads (Variant 56, Variant AA, Variant FF, and Variant GG) wereselected for animal trials and scaled up for 30 L fermentations toproduce at least 5 g of each protein. Based on activity and BradfordProtein analysis≧than 16 g of protein for each variant was produced.Recovered samples were lyophilized, then resuspended and heat treated tokill potential microbial growth and then re-lyophilized. These samples(Table 14) were used for animal trials. Along with the four selectedvariants, a small sample (15-50 mg) of the other SGF labile variants(Table 12) were used for bench scale evaluation.

TABLE 14 Specifications of samples used for animal trials and for benchscale evaluation. Variant Solid (g) Protein (g) Units Activity 56 (SEQID NO: 30) 106.2 8.34 1.37 × 10⁷ AA (SEQ ID NO: 28) 98.9 10.9 1.72 × 10⁷FF (SEQ ID NO: 24) 132.0 14.2 2.16 × 10⁷ GG (SEQ ID NO: 26) 109.6 17.43.09 × 10⁷ Variant Protein (mg) BB (SEQ ID NO: 32) 30 DD (SEQ ID NO: 36)50 CC (SEQ ID NO: 34) 20 EE (SEQ ID NO: 38) 40 HH (SEQ ID NO: 40) 15 SEQID NO: 2 30 The lyophilized product was quantified by both activity andBradford Protein Assay.MethodsGrowth, Induction, and PurificationE. coli Phytase Expression (2 L Scale)

All phytase variants, except of those in the glycosylation studies, wereexpressed in E. coli; strain PHY635 (phy-strain; created by making E.coli strain CU1867 (ATCC 47092; appA deficient) Rec A- through P1 phagetransduction). Starting cultures of the phytase variants were grown in 5mL LBcarb100 at 37 C for ˜18 hrs. 2 L of LBcarb100 were inoculated withthe overnight starting cultures, culture was induced with 1 mM IPTG whenthe culture reached ˜OD₆₀₀ 0.5. After 24 hrs of induction, the cultureswere harvested by pelleting the culture by centrifugation (Sorvall RC 5CPlus Centrifuge; SLC-4000 rotor, 7000 RPM; 9220 RCF) for 20 minutes. Thecells were resuspended in 50 mM Tris pH 8.0 and lysed utilizing themicrofluidizer (Microfluidics Model 11OL). To remove cellular debris thewhole cell lysate was centrifuged (Sorvall RC 5C Plus; F13S-14X50 Rotor,12500 RPM; 25642 RCF) for 30 minutes. The clear lysate was sterilefiltered and the phytase protein was purified across a HiisTrap FF 5 mLcolumn on an AKTA FPLC. Phytase was eluted off with a 2 M Immidizole, 50mM Tris pH 8.0 gradient. Fractions were tested for activity on 100 uMDiFMUP, 100 mM Na-Acetate (pH 5.5) and SDS-gel analysis for proteinpurity.

Pichia Pastoris Phytase Expression (1 L Scale)

The characterization of the final lead phytase variants(glycosylation-plus and glycosylation-minus versions of Variants AA-HHand Variant 56), along with the glycosylation-minus versions of SEQ IDNO:2 (Variants GLY1-GLY4) were expressed in Pichia pastoris (X-33).Starting cultures of the phytase variants were grown in 10 mL BMGYzeo100at 30° C. for ˜18 hrs (˜OD₆₀₀15-20), cells were pelleted and resuspendedin 10 mL MES* medium with 0.5% MeOH. 1 L of MES* medium with 0.5% MeOHwas inoculated with the starting culture and incubated for three-fourdays at 30° C. (5 mL MeOH added every 24 hrs for protein induction). Thesecreted protein separated from the cell mass by centrifugation (SorvallRC 5C Plus Centrifuge; SLC-4000 rotor, 7000 RPM; 9220 RCF), concentratedand buffer exchanged (100 mM Na Acetate pH 5.5) using the MiniKrosTangential Flow Separation Module (SpectrumLabs M215-600-01P). Toimprove purity the sample was passed across a HiTrap™ Q FF 5 mL columnon an AKTA FPLC. Phytase was eluted off with a 1 M NaCl, 100 mMNa-Acetate pH 5.5 gradient. Fractions were tested for activity on 100 uMDiFMUP, 100 mM Na-Acetate (pH 5.5) and SDS-gel analysis for proteinpurity.

Phytase Characterization

Protein Thermotolerance

Differential Scanning Calorimetry (DSC)—

Protein melting temperature (Tm) of the Pichia expressed phytasevariants were determined by utilizing the Applied Thermodynamics N-DSCII. Protein samples (˜1.0 mg/mL) were dialyzed in 100 mM Citrate pH 5.5,loaded into the test chamber (600 uL) and compared to the control sample(600 ul of 100 mM Citrate pH 5.5) and scanned from 60 to 100° C. andback to 60° C. (to assess protein refolding).

Modified Tm Determination—

Quick thermotolerance tool developed to evaluate whole cell lysate andnon purified protein samples during the preliminary characterization tocompare thermotolerance of SGF labile phytases to the parental phytase(SEQ ID NO:2). The protein sample was arrayed across a row on a 96 wellPCR plate (20 uL per well) and heat treated across a gradient (60-80°C.) on the PCR machine for 30 minutes. Heat treated protein samples (10uL) were mixed with fluorescence substrate (190 uL of 100 uM DiFMUP, 50mM Na-Acetate pH 5.5) measuring florescence change (EX360 nm/EM465 nm)over a five minute time course. The temperature at which 50% activityremained was compared to the parental phytase (SEQ ID NO:2) performance(50% activity temperature).

SGF Assay (Modified-Scaled Down)

Multiple mini reactions, quenched at different time points wereestablished to determine SGF lability of phytase molecule. As a controlT-0 reference, a pre-quenched SGF reaction was also run similar to theactual experiment. Ten uL of the protein sample was incubated in 50 uLof pH 1.2 SGF (2 mg/mL NaCl, 7 uL/mL concentrated HCl) with pepsin(dosed at 0.15, 0.30, and 0.75 mg/mL) over a time course at 37° C. Thereaction was quenched by adding 10 uL of pH 10.0, 200 mM Na-Carbonatebuffer (this step was performed prior to adding protein sample for theT-0 reference).

SGF SDS Gel Analysis—

Removed 20 uL of the quenched SGF reaction and mixed with 210 uL SDSsample buffer, boiled for 10 minutes, and loaded 15 uL onto a Tris-GlySDS Page gel. Applied 180V, 250 mA, through SDS sample running bufferfor ˜1 hour, or until complete.

SGF Activity Analysis—

Removed 10 uL of the quenched SGF reaction and mixed with substrate (190uL of 100 uM DiFMUP, 50 mM Na-Acetate pH 5.5) measuring florescencechange (EX360 nm/EM465 nm) over a five minute time course.

SGF Assay (Adapted from the United States Pharmacopeia 24, 2000.Simulated Gastric Fluid, TS, In The National Formulary 19; Board ofTrustees, Eds.; United States Pharmacopeial Convention, Inc., Rockville,Md., p. 2235)

Incubate 50 uL of 5 mg/mL phytase in pre-heated 37° C. 950 uL SGF (2mg/mL NaCl, titrated to pH 1.2 with HCl) with 10 U pepsin/ug testprotein (760 ug/mL SGF) over a 10 minute time course at 37° C. Timepoints were taken by removing 50 uL of reaction and mixing with 50 uLtermination solution (200 mM Na-Carbonate, pH 10.0). Time points(terminated samples) were kept on ice until assay was complete and readyfor analysis (in compliance with SGF SDS Gel Analysis and SGF ActivityAnalysis outlined under SGF Assay (Modified-Scaled Down)).

Phytase Specific Activity Analysis

Phytase samples (50 uL) were assayed for relative activity in pre heated37° C., 950 uL, 4.0 mM Phytate, 100 mM acetic acid, titrated to pH 4.5with NaOH. Reaction was quenched by removing 50 uL reaction and mixingwith the 50 uL color/stop solution (20 mM Ammonium molybdate/5 mMAmmonium vanadate/10% Nitric acid solution). After 10 minute colordevelopment time points were measure at 415 nm and results were plottedagainst time. The reaction rate was compared to the phosphate standardto determine relative rate.

The specific activity was determined by calculating relative rated basedon protein concentration. Protein concentration was determined by 260nm/280 nm analysis (1A OD₂₈₀ correlates to 0.93 mg/mL). A secondarycomparison was performed by loading equal phytase activities on SDS geland quantifying protein band intensities using GelPro gel densiotometryanalysis to compare activity relationship of phytase leads and theparental phytase (SEQ ID NO:2).

Phytase pH Profile Analysis

Same as the above specific activity protocol, except substrate wasmodified with a broader buffer capacity (pH 2-6). Substrate: 4 mMPhytate, 80 mM Malic acid, 80 mM Formic acid, and 80 mM Na-Acetatetitrated to different pH (2, 2.5, 3, 4, 5, and 6 pH Units). Relativerated for each variant were compared to the activity optimum which waspH 4.

Materials:

SGF Assay

Pepsin from porcine stomach mucosa (Sigma P-6887)

HCl (Fisher UN1789)

Sodium Chloride (Fisher S-271-1)

6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) (Invitrogen-D22068)

4-20% Tris-Glycine SDS PAGE Gels (Invitrogen EC60255BOX)

Novex Tris-Glycine SDS Sample Buffer (InvitrogenNovex LC2676)

Novex SDS Running Buffer (Invitrogen LC2675)

Simply Blue™ SafeStain (Invitrogen LC6065)

Phytase Specific Activity Analysis

Dodecasodium phytate from rice (Sigma P-3168)

Ammonium metavanadate (Acros Organics 194910500)

Ammonium molybdate (Sigma A-7302)

Potassium Phosphate, dibasic (Fisher P288-500)

70% Nitric Acid (Sigma 380091)

25% Ammonium Solution (Atlas Chemical AA-3060)

Protein Purification

HisTrap™ FF 5 mL Ni-Sepharose Column (GE Healthcare 17-5255-01)

HiTrap™ Q FF 5 mL Anion Exchange Column (GE Healthcare 17-5156-01)

Immidizole (Sigma I-0125)

A number of embodiments as provided herein have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope as provided herein.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. An isolated, synthetic, or recombinant variantpolypeptide of the amino acid sequence of SEQ ID NO:2, wherein thevariant polypeptide is an amino acid sequence at least 95%, identical tothe amino acid sequence as set forth in SEQ ID NO:2, and at least oneamino acid mutation selected from the group consisting of: T48F; T48H;T48I; T48K; T48L; T48M; T48V; T48W; T48Y; L50W; M51A; M51G; M51L; G67A;Y79H; Y79N; Y79S; Y79W; Q86H; P100A; S102A; S102Y; I107H; I107P; I108A;I108Q; I108R; I108S; I108Y; A109V; E113P; L126R; Q137F; Q137L; Q137V;Q137Y; D139Y; P145L; L146R; L146T; F147Y; N148K; N148M; N148R; P149L;P149N; L150T; L150Y; K151H; K151P; C155Y; L157C; L157P; V162L; V162T;T163P; L167S; G171M; G171S; S173G; S173H; S173V; I174F; I174P; V191A;L192F; F194L; S197G; S211H; L216T; P217D; P217G; P217L; P217S; S218I;S218Y; A232P; L235I; A236H; A236T; L244S; Q246W; Q247H; A248L; A248T;P254S; G257A; G257R; H263P; W265L; N266P; L269I; L269T; H272W; A274F;A274I; A274L; A274T; A274V; Q275H; T282H; T291V; T291W; Q309P; P343E;P343I; P343L; P343N; P343R; P343V; N348K; N348W; G353C; Q377R; L379S;L379V; Q381S; S389H; S389V; G395E; G395I; G395L; G395Q; G395T; V422M;I427G; I427S; I427T; and A429P, wherein the variant polypeptide hasphytase activity and have decreased gastric stability when compared tothe parent polypeptide of the amino acid sequence of SEQ ID NO:2.
 2. Aprotein preparation comprising the polypeptide of claim 1, wherein theprotein preparation comprises a liquid, a slurry, or a powder.
 3. Amethod for hydrolyzing an inositol-hexaphosphate to inositol andinorganic phosphate comprising: (a) providing the polypeptide of claim1; (b) providing a composition comprising an inositol-hexaphosphate; and(c) contacting the polypeptide of (a) with the composition of (b) underconditions wherein the polypeptide hydrolyzes the inositol-hexaphosphateto produce to inositol and inorganic phosphate.
 4. A method for oildegumming comprising: (a) providing the polypeptide of claim 1; (b)providing a composition comprising an oil; and (c) contacting thepolypeptide of (a) and the oil of (b) under conditions wherein thepolypeptide can cleave an inositol-inorganic phosphate linkage, therebydegumming the oil.
 5. A food, a feed, a food supplement, or a feedsupplement for an animal comprising the polypeptide of claim
 1. 6.A-pellet comprising the polypeptide of claim
 1. 7. A soybean mealcomprising the polypeptide of claim
 1. 8. A method for processing ofcorn and sorghum kernels comprising (a) providing the polypeptide ofclaim 1; (b) providing a composition comprising a corn steep liquor or asorghum steep liquor; and (c) contacting the polypeptide of (a) and thecomposition of (b) under conditions wherein the polypeptide can cleavean inositol-inorganic phosphate linkage.
 9. A composition comprising thepolypeptide of claim 1, wherein the composition comprises a secondenzyme.
 10. The composition of claim 9, wherein the second enzyme isselected from a second phytase, a xylanase, a cellulase, a glucanase, apullanase, a mannanase, a protease, a lipase, a beta-glucosidase, acelloiohydroase, an amylase, and any combination thereof.
 11. The foodof claim 5, wherein the food is a bread dough.
 12. The feed of claim 5,wherein the animal is livestock.
 13. The feed of claim 12, wherein thelivestock is selected from: poultry, pigs, cattle, sheep, goats, andfish.